The importance of diagnostic for deadly infectious diseases, particularly in resource-deprived settings, cannot be understated. Diagnostic is most important to determine the type and level of medical intervention required, particularly for diseases where onset symptoms are similar, and also to understand the extent and spread of an outbreak.
The ideal diagnostic kit
While RT-PCR and ELISAs are the most common and versatile tools in the diagnostic toolbox, the complexity and time required by these procedures, together with the need to preserve many of their components cold, represent a problem. Ideally, what is needed is a system that does not require cold transportation and storage, is easy to operate with minimal exposure to the infectious samples, provides results in no more than 30 minutes, and could be used to distinguish between multiple pathogens at the same time. Oh, and it should also be accurate, sensitive, and cheap. Wishful thinking?
Maybe not. Several companies and organizations have taken up the challenge and applied their technical expertise to the field, coming up with clever solutions. Here we cover the strategy presented by scientists from Beckton Dickinson and from USA and African universities in the journal Science Translational Medicine, based on Raman spectroscopy.
A bit of physics
The technology is based on SERS nanotags, where SERS stands for surface-enhanced Raman spectroscopy (and nanotags for nanoparticle tags!). Raman spectroscopy allows identification of different molecules as it provides an accurate structural fingerprint of chemical matter. In Raman spectroscopy, a molecule is hit by a laser in the visible, near infrared, or near ultraviolet range. The laser light interacts with the molecular vibrations of the system, which in turns causes the laser photons to be shifted. It is this shift that gives information on the vibrational, rotational, and other low frequency modes of the systems, which, being different for every molecule, in turn represent the molecular fingerprint of the chemical entity.
The sphere
In their system, the authors use three different Raman dyes (giving different and unique Raman fingerprints) to distinguish between three different pathogens: Ebola virus (EBOV), Lassa virus (LASV), and the malaria parasite.
Each dye (or Raman reporter) is placed all around 60 nm gold particles, which have the ability to increase the Raman signal by 4-8 orders of magnitude, allowing the signal to be detected by less sensitive (and less expensive) equipment.
Next, these dye-covered nanoparticles are further enveloped into a 20-30 nm silica shell, which has two functions: preventing the dye from coming off the gold particle, and blocking non specific Raman signals coming from the sample tested.
Once stimulated by a 785 nm laser source, the SERS signal is read in the near infrared (820 to 914 nm), where clinical samples like blood create minimal interference. And when the three silica-dye-gold nanoparticles are tested in the system, they give clear and different spectra allowing accurate recognition of the three different Raman reporters.
The sandwich
This system now needs to be conjugated with elements that will enable recognition of the different pathogens. Cleverly, the silica gel in the outer shell incorporates sulfhydryl groups, which allow conjugation of pathogen-specific antibodies. The antibodies recognize the malaria biomarker HRP2, the EBOV matrix VP40, or the LASV nucleoprotein. The same antibodies are also conjugated to magnetic microparticles that will be added into the mix.
Now all we have to add is the blood sample (just 45 μl are sufficient) and lysis buffer, close the lid, and let all the components mix by placing the tube on a rotating wheel for 25 minutes. The virus contained in the sample will be recognized by the antibodies on the SERS nanotag, as well as by the antibodies on the magnetic particles, generating a molecular sandwich with the recognized pathogen/antigen in the middle. At the end of incubation time, a small magnet is applied to the outside of the tube for 5 minutes, causing the magnetic particles to bring all the sandwiches to the same spot, where they can be hit by the laser to measure their fingerprint. If the sample was positive for EBOV, the fingerprint will be the one of the dye conjugated to anti-EBOV antibodies. If the sample was positive for, say, EBOV and malaria, the two fingerprints will be detected. Not only this: the measured signal is proportional to the concentration of the target, providing quantitative and not only qualitative information.
In addition to specificity and speed, the assay just requires pipetting whole blood into a tube already containing all the assay components, without any sample processing or the need to re-open tubes containing infectious material. This constitutes a great advantage as it limits the need for specialist training and the risk of pathogen exposure. But does it work?
Testing the system
The authors tested the system in three different and equally important applications: 1) pure virus, to test and calibrate the assay; 2) fresh or frozen blood samples from non-human primate (NHP); and 3) frozen human blood samples from previous epidemics.
Recombinant antigens and virus-spike blood samples showed good analytical sensitivity for all samples, especially for the malaria marker HRP2. EBOV was recognized at from 1×105 infectious units/ml and LSV from 5×106 infectious units/ml, placing the analytical sensitivity of SERS technology in the same range of ELISAs. Importantly, negative samples came up negative, confirming specificity. Also, when multiple antigens/viruses were mixed in the same tube containing multiple probes, the system showed high specificity and no substantial cross-reactivity.
NHP fresh blood samples performed equally well, and in a similar range as recombinant antigens.
586 frozen clinical specimens from Senegal and Guinea banks were also tested, and the results for EBOV and malaria compared with gold standard reference methods. No LASV positive samples were collected, so only LASV negative samples could be used to test the specificity of this kit component.
Besides testing the specificity, sensitivity, and accuracy of the system, this trial also allowed to assess the effectiveness of this diagnostic method in facilities with limited resources, and of reconstituting lyophilized reagents ready inside the tubes by direct addition of blood samples and lysis buffer. The sensitivity for EBOV detection was 90% at a specificity of 97.9%, for a total accuracy of 96.6%. For malaria, sensitivity was 100%, specificity 99.6%, and accuracy 99.7%. All LASV were negative.
Outlook
Whether the costs and the scalability of this setting are compatible with diagnostic in the context of an outbreak will have to be determined in the field; however, the data presented are indicative of a clean and accurate system with similar sensitivity to current gold standards, and with the additional advantages of limited exposure to dangerous pathogens and reduced need for extensive training, long assay times, clean settings (as for PCR), and a cold chain to transport and store reagents.
The application of the same principles to other pathogens could also expand the multiplex capability of the system, making it an invaluable piece of diagnostic kit.
