Biology subjects

Lucchi, N. W.

Publications and source records attributed to Lucchi, N. W..

4 recordsLinked to original sources

bioRxiv2025

Assessment of different techniques and markers to distinguish recrudescence from new infection in an antimalarial therapeutic efficacy study conducted in Rwanda

IntroductionAccurate estimation of antimalarial drug efficacy against P. falciparum requires PCR correction to distinguish recrudescence from new infection in recurrent infections. Genotyping length polymorphic or SNP-rich markers, and various decision algorithms based on match counting or probabilistic approaches may be used for this purpose. In this study, we compared several markers and decision algorithms using samples collected in a therapeutic efficacy study conducted in Rwanda to identify the most suitable and robust approaches for PCR correction. MethodsWe optimized nested PCR assays to genotype four microsatellites and assessed their sensitivity in detecting minority clones with laboratory parasite strain mixtures. We analyzed patient samples by capillary electrophoresis and amplicon deep sequencing and assessed the diversity and allelic frequency of msp1, msp2, glurp, microsatellites (Poly-, PfPK2, TA40, TA81), and SNP-rich markers (ama1-D2, ama1-D3, cpmp, cpp, csp). We then classified the samples into recrudescence or new infection based on different marker combinations (msp1/msp2/glurp, msp1/msp2/microsatellite, and SNP-rich markers) and three decision algorithms (WHO algorithm, 2/3 algorithm, and a Bayesian approach), and compared our results with previously published findings. ResultsAmong microsatellites, TA40 and PfPK2 had the highest sensitivity in detecting minority clones; however, their intra-assay reproducibility was found to be limited. The 3D7 allelic family of the msp2 gene, glurp and SNP-rich markers had the highest genetic diversity. In distinguishing recrudescence from new infection, the WHO algorithm identified the fewest recrudescences across all marker combinations. Conversely, the 2/3 algorithm identified the highest number of recrudescences, and the algorithm based on Bayesian statistics yielded intermediate results. We observed the least variability in classification results among the different algorithms for the SNP-rich markers ama1-D2/ama1-D3/cpmp combination. In our study, replacing glurp with one of the four analyzed microsatellites or using SNP-rich markers did not significantly alter the final number of recrudescent infections. InterpretationOur findings emphasize the importance of using assays with high sensitivity in detecting minority clones, and markers with high diversity and low allelic frequency for accurate PCR correction. The SNP-rich markers method yielded the most consistent results in detecting recrudescences, regardless of the decision algorithm used. Therefore, it holds great potential for performing reliable PCR correction. Further evaluation of decision algorithms based on probabilistic approaches, compared to match-counting methods, is essential to ensure the accuracy and consistency of PCR-corrected drug efficacy estimations.

molecular biology↗

bioRxiv2024

Trends of Plasmodium falciparum molecular markers associated with resistance to artemisinins and reduced susceptibility to lumefantrine in Mainland Tanzania from 2016 to 2021

BackgroundTherapeutic efficacy studies (TESs) and detection of molecular markers of drug resistance are recommended by the World Health Organization (WHO) to monitor the efficacy of artemis inin combination therapy (ACT). This study assessed the trends of molecular markers of artemis inin resistance and/or reduced susceptibility to lumefantrine using samples collected in TES conducted in Mainland Tanzania from 2016 to 2021. MethodsA total of 2,015 samples were collected during TES of artemether-lumefantrine at eight sentinel sites (in Kigoma, Mbeya, Morogoro, Mtwara, Mwanza, Pwani, Tabora, and Tanga regions) between 2016 and 2021. Photo-induced electron transfer polymerase chain reaction (PET-PCR) was used to confirm presence of malaria parasites before capillary sequencing, which targeted two genes: Plasmodium falciparum kelch 13 propeller domain (k13) and P. falciparum multidrug resistance 1 (pfmdr1). ResultsSequencing success was [≥]87.8%, and 1,724/1,769 (97.5%) k13 wild-type samples were detected. Thirty-seven (2.1%) samples had synonymous mutations and only eight (0.4%) had non-synonymous mutations in the k13 gene; seven of these were not validated by WHO as molecular markers of resistance (I416V, E433D, R471S, P475S, A578S, and Q613E). One sample from Morogoro in 2020 had a k13 R622I mutation, which is a validated marker of artemisinin partial resistance. For pfmdr1, all except two samples carried N86 (wild-type), while mutations at Y184F increased from 33.9% in 2016 to about 60.5% in 2021, and only four samples (0.2%) had D1246Y mutations. pfmdr1 haplotypes were reported in 1,711 samples, with 985 (57.6%) NYD, 720 (42.1%) NFD, and six (0.4%) carrying minor haplotypes (three with NYY, 0.2%; YFD in two, 0.1%; and NFY in one sample, 0.1%). Between 2016 and 2021, NYD decreased from 66.1% to 45.2%, while NFD increased from 38.5% to 54.7%. ConclusionThis is the first report of the R622I (k13 validated mutation) in Tanzania. N86 and D1246 were nearly fixed, while increases in Y184F mutations and NFD haplotype were observed between 2016 and 2021. Despite the reports of ART-R in Rwanda and Uganda, this study did not report any other validated mutations in these study sites in Tanzania apart from R622I suggesting that intensified surveillance is urgently needed to monitor trends of drug resistance markers and their impact on the performance of ACTs.

molecular biology↗

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↗

bioRxiv2018

The ER chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites.

The vast majority of malaria mortality is attributed to one parasite species: Plasmodium falciparum. Asexual replication of the parasite within the red blood cell is responsible for the pathology of the disease. In Plasmodium, the endoplasmic reticulum (ER) is a central hub for protein folding and trafficking as well as stress response pathways. In this study, we tested the role of an uncharacterized ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localizes to the ER and is essential for asexual growth, specifically required for proper development of schizonts. PfGRP170 is essential for surviving heat shock, suggesting a critical role in cellular stress response. The data demonstrate that PfGRP170 interacts with the Plasmodium orthologue of the ER chaperone, BiP. Finally, we found that loss of PfGRP170 function leads to the activation of the Plasmodium eIF2 kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway.

cell biology↗