Making Specters of the Vectors

The recent SARS-CoV-2 pandemic drove an unprecedented demand for molecular diagnostic methods that could be used in environments outside the traditional clinical laboratory. Of the available methods, loop-mediated isothermal amplification (LAMP) immediately attracted interest because it can be performed in under 30 minutes at a single temperature – without requiring instrumentation.

Adding to the method’s simplicity and flexibility, LAMP tests can be assessed by visual inspection. In colorimetric LAMP, a positive result is reflected by a color-change; colorimetric indicators interact with the byproducts of extensive DNA synthesis (1). This combination of a simple, fast, and easily interpreted molecular diagnostic test made LAMP an optimal candidate for at-home testing – and colorimetric LAMP enabled the first-ever molecular diagnostic test approved for at-home use.

Although the pandemic catapulted LAMP’s popularity as a diagnostic tool, a number of fields – from agriculture to public health – can benefit from a molecular test that easily identifies DNA and RNA targets. For example, many infectious diseases are spread via insect vectors, and the surveillance of infectious disease in those vectors can contribute to a better understanding of disease propagation and reservoir geography, helping inform appropriate public health measures. With LAMP, these insect vectors can be tested in the field or on-site – allowing for real-time data collection and surveillance in regions that lack easy access to laboratory facilities.

Vector-borne disease detection and monitoring
 

Early work with LAMP, which was first described in 2000, has focused on neglected tropical diseases, particularly filarial parasite targets in which the diagnostic needs are high, but where the typical testing infrastructure is not in place (2). These ongoing efforts include not only testing human patients for infection, but also surveilling the vector population to inform epidemiology and control efforts. Here, LAMP’s ability to tolerate crude and unpurified samples has proven to be a critical factor in its practical application to field and point-of-need tests in which sample processing must be kept as simple as possible.

For instance, researchers have used LAMP to identify the filarial parasite Onchocerca volvulus in black flies, which causes Onchocerciasis, commonly known as “river blindness.” Pools of 50–200 black flies were crushed in a tube and DNA was extracted by simple boiling then added directly to colorimetric LAMP reactions. Onchocerca volvulus genomic DNA could then be detected down to 0.01 ng in a pool of 100 insects by visual inspection of the reaction color (3).

A colorimetric LAMP assay has also recently been validated for the identification of Mansonella perstans – a filarial parasitic nematode – which is one of the causes of Mansonellosis (4). Mansonellosis, which may down-regulate immune responses (enhancing susceptibility to other infections, such as tuberculosis and malaria, and negatively affecting the efficacy of vaccines) is the least studied of the filarial diseases, with few alternative reliable and accessible methods of diagnosis (5,6,7). At present, no other immunoassays have been developed, and available PCR-based methods rely on trained personnel and relatively expensive equipment that restrict widespread adoption.

In addition to detecting parasites responsible for vector-borne diseases, LAMP has also demonstrated promise in identifying insect vectors. Of these, mosquitoes are the most prominent and result in the most infections and death worldwide. Mosquitoes are responsible for the transmission of numerous parasitic diseases, as well as arboviruses, such as Zika, West Nile, Chikungunya, and more (8).  As different species of mosquito serve as hosts for different pathogens, identifying the specific type of mosquito can provide value to surveillance efforts. LAMP tests have been demonstrated for mosquito speciation directly from larvae, enabling the identification of problematic species (for example, Aedes aegypti and albopictus) potentially at ports of entry or in control programs (9). Such LAMP tests have been described for crushed mosquitos or pools in laboratory and field settings. 

Assessing vector-borne disease control efforts

Alongside vector-borne disease detection and monitoring, LAMP-assisted surveillance has proven to be a powerful tool for assessing the efficacy of vector-borne disease control efforts. For example, over the past two decades, there have been programs in place to control onchocerciasis with the mass drug administration (MDA) of ivermectin, which involves administering the drug to an entire community regardless of whether individuals are infected or not. The impact of these programs has been variable, and careful monitoring of infection in humans and vectors is now needed to evaluate where MDA programs are – or are not – successful.

However, the effectiveness of MDA programs has been difficult to evaluate through conventional microscopy-based methods of diagnosing onchocerciasis. These methods struggle to differentiate the filarial nematodes responsible for onchocerciasis from co-endemic infection by other parasitic filarial nematodes, including Loa loa, commonly known as the African eye worm, and Mansonella perstans. Where conventional methods fail, LAMP assays that have been developed for Loa loa, Mansonella perstans, and Onchocerca volvulus, respectively, could prove vital to the surveillance efforts used by global health programs aimed at achieving the elimination of onchocerciasis (10).

Another exemplar of LAMP’s utility in assessing vector-borne disease control efforts is the World Mosquito Program, which uses a method of controlling mosquito-borne diseases that involves the release of mosquitoes intentionally infected with a commensal bacteria called Wolbachia. This bacteria makes it harder for viruses – including infectious viruses, such as Zika and yellow fever – to reproduce in mosquitos. Programs that successfully spread the virus-suppressing commensal bacteria from the released population into native mosquitoes reduce the risk of mosquito-borne diseases in that region. The World Mosquito Program has been able to do surveillance of release trials in Australia, Brazil, and across Southeast Asia using LAMP tests targeting the introduced wMel Wolbachia bacterial endosymbiont, so that researchers could better understand the dynamics of the release programs and Wolbachia’s stability in mosquito populations over time (11). 

The growing need for surveillance
 

With climate change exacerbating the risk of vector-borne diseases, the need for surveillance of insect vectors and vector-borne diseases is likely to rise worldwide. In 2023, the US saw locally acquired malaria cases for the first time in 20 years and Spain observed an increase in dengue transmission, indicating a spread of vectors to new, warmer areas. And though mosquito-borne diseases have generally been seen as a problem for the Global South, surveillance efforts for Eastern Equine Encephalitis and West Nile viruses have established importance in areas where those diseases are endemic in North America (12).

Beyond flies and mosquitoes, surveillance efforts in North America and Europe have focused on tick-borne diseases, with a rising incidence of bacterial (Borrelia, Anaplasma, Rickettsia), parasitic (Babesia), and viral (tick-borne encephalitis virus, Powhassan) infections. As the tick host population spreads to new environments in which ticks had previously been unable to survive – facilitated by climate change – awareness and the need for identifying these pathogens is increasing; LAMP can serve as first-line detection. Although ticks are more difficult to easily process in field-based testing, they can be crushed or sufficiently lysed without full extraction protocols to provide sample material to a colorimetric or other simple LAMP reaction (13,14).

Evolving applications and innovations for LAMP
 

The increased availability and application of powerful molecular diagnostic tools will have a significant impact on public health worldwide. Moreover, innovations to platforms such as LAMP will further expand the reach of molecular methods outside the laboratory where this impact can be maximized. 

Already, the diverse range of mosquito LAMP assays has resulted in a wide range of workflows and platform development, including a simple by-eye analysis of color change to more quantitative measurements using smartphones, or simple devices to measure fluorescence or lateral flow strip analysis. Maximizing portability and simplicity, LAMP has also been conducted in full paper-based systems in which sample treatment, incubation, and detection all occur within a disposable, low-cost paper device.

By enabling testing directly from samples in the field or at the point of need, LAMP can bring rapid and cheap identification of molecular targets to new environments. Insect vectors present a continual and growing threat, and increased use of LAMP will help expand our ability to monitor them and prevent the dangerous diseases they will spread to new populations.