Louzada, J. | bioRxiv Science

bioRxiv2025

Emergence of Artemisinin-based Combination Therapy Resistance Markers in Plasmodium falciparum from the Brazilian Tri-Border Region of the Guiana Shield

Plasmodium falciparum malaria remains a public health concern in the Brazilian Amazon, particularly among mobile and remote populations. Antimalarial drug resistance threatens elimination efforts, particularly in the Guiana Shield, which has historically been linked to the spread of resistant parasites. We analyzed 99 P. falciparum isolates collected in Roraima State, northern Brazil (2016-2020), using targeted deep sequencing. Variants were assessed in pfcrt, pfmdr1, and pfk13, and copy number variation in pfpm2 and pfpm3, including detection of the pfpm2/3 hybrid. High coverage enabled the detection of both dominant and minority alleles. Among the 99 isolates, the pfmdr1-NFD haplotype was found in 6% (6/99, all likely originating from Venezuela), pfcrt-C350R in 7% (7/99), and pfpm2 and pfpm3 amplifications in 5% (5/94) and 6% (6/97), respectively, and the pfpm2/3 hybrid in 4% (4/99). No validated or candidate pfk13 mutations associated with artemisinin partial resistance were detected. This study provides the first report of the pfpm2/3 hybrid and the pfmdr1-NFD haplotype in Brazil, and confirms the presence of pfcrt-C350R in isolates from the tri-border area. These variants, associated with reduced susceptibility to lumefantrine and piperaquine, suggest that parasites in northern Brazil already carry resistance markers that are prevalent in neighboring countries. These results highlight the need for continued genomic surveillance to detect early warning signals, guide treatment policies, and contain the spread of parasites with reduced susceptibility to ACT partner drugs.

microbiology↗

bioRxiv2018

Field evaluation of malachite green loop-mediated isothermal amplification as a malaria parasite detection tool in a health post in Roraima state, Brazil

Malaria is a debilitating parasitic disease that causes significant morbidity and mortality. Microscopic detection of parasites is currently the \"gold standard\" diagnostic. This technique is limited in its ability to detect low-density infections, is time consuming, and requires a highly trained microscopist. Malaria epidemiological surveillance studies especially aimed at the detection of low-density infection and asymptomatic cases will require more sensitive and user-friendly tools. We have shown previously that the molecular-based, colorimetric malachite green loop-mediated isothermal amplification (MG-LAMP) assay is a valuable tool for diagnosing malaria infection in a laboratory setting. In this study, we field evaluated this assay in a malaria diagnostic post in Roraima, Brazil. We prospectively collected 91 patient samples and performed microscopy, MG-LAMP, and real-time PCR (PET-PCR) to detect Plasmodium infection. Two independent readers were used to score the MG-LAMP tests to assess whether the sample was positive (blue/green) or negative (clear). There was 100% agreement between the two readers (Kappa=1). All tests detected 33 positive samples, but both the MG-LAMP and PET-PCR detected 6 and 7 more positive samples, respectively. The PET-PCR assay detected 6 mixed infections (defined as infection with both P. falciparum and P. vivax) while microscopy detected one and MG-LAMP detected two of these mixed infections. Microscopy did not detect any Plasmodium infection in 26 of the enrolled asymptomatic cases while MG-LAMP detected five and PET-PCR assay three positive cases. Overall, MG-LAMP provided a simpler and user-friendly molecular method for malaria diagnosis that is more sensitive than microscopy. Additionally, MG- LAMP has the capacity to test 38 samples per run (one hour), allowing for the screening of large number of samples which is appealing when large-scale studies are necessary e.g. in community surveillance studies. The current MG-LAMP assay was limited in its ability to detect mixed infection when compared to the PET-PCR, but otherwise proved to be a powerful tool for malaria parasite detection in the field and opens new perspectives in the implementation of surveillance studies in malaria elimination campaigns.

epidemiology↗