Colorimetric LAMP Test Spots Hidden Malaria Infections in the Peruvian Amazon

Malaria is slipping quietly through the net in the Peruvian Amazon, and a new study suggests that a simple, low-cost molecular test could help catch it. In research published in PLOS Neglected Tropical Diseases, a team led by Oscar Nolasco and Christopher Delgado-Ratto evaluated a colorimetric loop-mediated isothermal amplification assay, or cLAMP, for detecting the two malaria parasites that circulate in the region, Plasmodium vivax and Plasmodium falciparum. The work, conducted on dried blood spots collected during routine surveillance, offers a practical route to finding the low-density infections that microscopy routinely misses and that standard PCR is often too expensive or logistically demanding to deploy at scale.

The challenge the researchers set out to address is familiar to every elimination program. As transmission falls, infected individuals increasingly carry very small numbers of parasites in their blood, often below the threshold that a microscopist examining a blood smear can reliably detect. These submicroscopic infections still contribute to onward transmission, because mosquitoes that feed on an infected person can pick up gametocytes even when the density of parasites in the peripheral blood is tiny. In settings such as the Amazon basin, where P. vivax in particular hides in the liver and re-emerges in relapses, missing these infections allows reservoirs of disease to persist in communities that appear, on paper, to be approaching elimination.

Loop-mediated isothermal amplification was developed as a molecular diagnostic that avoids the thermal cycling equipment required by conventional PCR. Instead of repeatedly heating and cooling a sample, LAMP uses a strand-displacing DNA polymerase and four to six specially designed primers that recognize six to eight distinct regions of the target genome. The reaction proceeds at a constant temperature, typically around 60 to 65 degrees Celsius, which can be maintained by a simple heating block or even a water bath. Amplification produces enormous quantities of pyrophosphate and protons as byproducts, and in a colorimetric formulation a pH-sensitive dye shifts color when the reaction succeeds, turning a positive result into something a technician can read with the naked eye.

That simplicity is what makes cLAMP attractive for field surveillance, but the technology has a well-known Achilles heel: sample preparation. DNA extraction remains the most technically demanding and contamination-prone step in any molecular assay, and the choice of extraction method can dominate overall performance. The Peruvian team confronted this directly by comparing two approaches on the same samples. The first was a mini spin column method, in which DNA is bound to a silica membrane, washed, and eluted in clean buffer, a process that concentrates and purifies genetic material but requires more reagents, equipment, and hands-on time. The second was a boil-and-spin method, in which a small piece of dried blood is simply heated in water or buffer to release DNA, then briefly centrifuged to pellet debris, leaving crude lysate ready for amplification.

The samples came from two complementary surveillance strategies that elimination programs run in parallel. Active case detection, abbreviated ACD, involves health workers visiting communities and screening people regardless of whether they feel ill, in order to flush out asymptomatic carriers. Passive case detection, or PCD, relies on patients who come to a clinic with fever or other symptoms. The study analyzed 574 dried blood spots from active detection and 431 from passive detection. Dried blood spots themselves are a surveillance workhorse: a finger-prick of blood spotted onto filter paper, dried, and stored, they are cheap to collect, stable at ambient temperatures for weeks to months, and far easier to transport from remote river communities to a laboratory than whole blood or slides.

To judge how well cLAMP performed, the researchers benchmarked it against two references. Real-time quantitative PCR, or qPCR, served as the molecular gold standard, while microscopy represented the current reality of most field diagnosis. The gap between the two was stark. In the active detection set, qPCR identified 125 positive samples, 117 of them P. vivax and 8 P. falciparum. Microscopy, run against that standard, achieved a sensitivity of only 57.6 percent, meaning it missed more than four in ten infections that were genuinely present. cLAMP with the crude boil-and-spin extraction did somewhat better, reaching 63.2 percent sensitivity, while cLAMP paired with the spin column extraction climbed to 82.4 percent, a dramatic improvement over the smear.

The passive detection samples told a related but distinct story. Here qPCR found 140 positives, comprising 129 P. vivax infections, 9 P. falciparum infections, and 2 mixed infections containing both species. Because symptomatic patients generally carry higher parasite densities, microscopy fared better in this group, achieving 72.9 percent sensitivity. cLAMP with boil-and-spin extraction reached 77.1 percent, roughly comparable to the smear, while the column-based extraction again pushed sensitivity to 82.1 percent. Across both surveillance modes and all diagnostic methods, specificity remained above 99 percent, indicating that false positives were vanishingly rare and that a positive result could be trusted as a true infection.

The pattern in these numbers carries a clear technical message. The amplification chemistry itself is sensitive enough, but its real-world performance is gated by the quality of the DNA template fed into it. Boil-and-spin extraction releases parasite DNA but also carries blood-derived inhibitors and leaves the template dilute, which hurts most precisely where sensitivity matters most: in the low-density, often asymptomatic infections found by active case detection. Column-based extraction cleans and concentrates the template, and the benefit shows up disproportionately in the ACD samples, where cLAMP with columns outperformed microscopy by nearly 25 percentage points. In passive surveillance, where parasite densities run higher, the extraction method mattered less because even crude lysate contained enough template for the reaction to succeed.

For program managers weighing options in low-transmission settings, the study frames a trade-off rather than a single verdict. Boil-and-spin cLAMP is cheaper, faster, and simpler, and it performs at least as well as microscopy, making it a plausible upgrade for laboratories that cannot sustain column-based workflows. Column-based cLAMP, meanwhile, delivers sensitivity approaching qPCR from a filter paper sample, without needing a thermocycler or fluorescent detection hardware, and it substantially outperforms the smear in exactly the active surveillance campaigns where finding hidden infections is the whole point. Because the readout is a visible color change, results do not require expensive instruments, and the isothermal reaction can run on basic laboratory equipment already common in regional health posts.

The authors conclude that cLAMP, particularly when combined with column-based DNA extraction, is a practical and sensitive complementary tool for malaria surveillance in elimination settings. As countries in the Amazon and beyond push toward the last mile of malaria elimination, the infections that matter are the ones nobody can see under a microscope, and tools that can find them cheaply, reliably, and close to the communities where they occur will determine whether the final stretch of the campaign succeeds. This study suggests that a color-changing tube, a filter paper spot, and a careful choice of extraction chemistry may be enough to bring molecular-grade detection out of the reference laboratory and into the field.

Subject of Research: Evaluation of colorimetric loop-mediated isothermal amplification for malaria detection from dried blood spots in the Peruvian Amazon

Article Title: Detection of Plasmodium vivax and Plasmodium falciparum using colorimetric loop-mediated isothermal amplification from dried blood spots collected in active and passive malaria surveillance in the Peruvian Amazon

Article References: Nolasco, O., Gómez, J., Rodríguez-Ferrucci, H., Gamboa, D., & Delgado-Ratto, C. (2026). Detection of Plasmodium vivax and Plasmodium falciparum using colorimetric loop-mediated isothermal amplification from dried blood spots collected in active and passive malaria surveillance in the Peruvian Amazon. PLOS Neglected Tropical Diseases, 20(10), e0014366. https://doi.org/10.1371/journal.pntd.0014366

Image Credits: AI Generated

DOI: 10.1371/journal.pntd.0014366

Keywords: malaria, Plasmodium vivax, Plasmodium falciparum, LAMP, colorimetric assay, dried blood spots, molecular diagnostics, active case detection, passive case detection, Peruvian Amazon, malaria elimination, DNA extraction