Spatiotemporal dynamics of Plasmodium falciparum histidine-rich protein 2 and 3 deletions in Peru.

Peru was the first country where pfhrp2 and pfhrp3 gene deletions were detected despite the fact that rapid diagnostics tests are not commonly used for confirmatory malaria diagnosis. This context provides a unique scenario to study the dynamics of pfhrp2 and pfhrp3 gene deletions without apparent RDTs selection pressure. In this study we characterized the presence of pfhrp2 and pfhrp3 genes on 325 P. falciparum samples collected in Iquitos and surrounding communities between 2011 and 2018 in order to understand the dynamics of gene deletion prevalence, potential associations with clinical symptomatology and parasite genetic background. P. falciparum presence was confirmed by microscopy and PCR of 18 s rRNA, pfmsp1 and pfmsp2. Gene deletions were assessed by amplification of exon1 and exon2 of pfhrp2 and pfhrp3 using gene specific PCRs. Confirmation of absence of HRP2 expression was assessed by ELISA of HRP2 and pLDH. Genotyping of 254 samples were performed using a panel of seven neutral microsatellite markers. Overall, pfhrp2 and pfhrp3 dual gene deletions were detected in 67% (217/324) parasite samples. Concordance between pfhrp2 deletion and negligible HRP2 protein levels was observed (Cohen's Kappa = 0.842). Prevalence of gene deletions was heterogeneous across study sites (adjusted p < 0.005) but there is an overall tendency towards increase through time in the prevalence of dual pfhrp2/3-deleted parasites between 2011 (14.3%) and 2016 (88.39%) stabilizing around 65% in 2018. Dual deletions increase was associated with dominance of a single new parasite haplotype (H8) which rapidly spread to all study sites during the 8 study years. Interestingly, participants infected with dual pfhrp2/3-deleted parasites had a significantly lower parasitemias than those without gene deletions in this cohort. Our study showed the increase of pfhrp2/3 deletions in the absence of RDTs pressure and a clonal replacement of circulating lines in the Peruvian Amazon basin. These results suggest that other factors linked to the pfhrp2/3 deletion provide a selective advantage over non-deleted strains and highlight the need for additional studies and continuing surveillance.

Drug resistance and population structure of Plasmodium falciparum and Plasmodium vivax in the Peruvian Amazon.

Malaria is a major health problem in Peru despite substantial progress achieved by the ongoing malaria elimination program. This study explored the population genetics of 63 Plasmodium falciparum and 170 P. vivax cases collected in the Peruvian Amazon Basin between 2015 and 2019. Microscopy and PCR were used for malaria detection and positive samples were genotyped at neutral and drug resistance-associated regions. The P. falciparum population exhibited a low nucleotide diversity (π = 0.02) whereas the P. vivax population presented a higher genetic diversity (π = 0.34). All P. falciparum samples (n = 63) carried chloroquine (CQ) resistant mutations on Pfcrt. Most P. falciparum samples (53 out of 54) carried sulfadoxine (SD) resistant mutations on Pfdhfr and Pfdhps. No evidence was found of artemisinin resistance mutations on kelch13. Population structure showed that a single cluster accounted for 93.4% of the P. falciparum samples whereas three clusters were found for P. vivax. Our study shows a low genetic diversity for both species with significant differences in genetic sub-structuring. The high prevalence of CQ-resistance mutations could be a result of indirect selection pressures driven by the P. vivax treatment scheme. These results could be useful for public health authorities to safeguard the progress that Peru has achieved towards malaria elimination.

Validation study of Boil & Spin Malachite Green Loop Mediated Isothermal Amplification (B&S MG-LAMP) versus microscopy for malaria detection in the Peruvian Amazon.

Malaria elimination efforts in Peru have dramatically reduced the incidence of cases in the Amazon Basin. To achieve the elimination, the detection of asymptomatic and submicroscopic carriers becomes a priority. Therefore, efforts should focus on tests sensitive enough to detect low-density parasitemia, deployable to resource-limited areas and affordable for large screening purposes. In this study, we assessed the performance of the Malachite-Green LAMP (MG-LAMP) using heat-treated DNA extraction (Boil & Spin; B&S MG-LAMP) on 283 whole blood samples collected from 9 different sites in Loreto, Peru and compared its performance to expert and field microscopy. A real-time PCR assay was used to quantify the parasite density. In addition, we explored a modified version of the B&S MG-LAMP for detection of submicroscopic infection in 500 samples and compared the turnaround time and cost of the MG-LAMP with microscopy. Compared to expert microscopy, the genus B&S MG-LAMP had a sensitivity of 99.4% (95%CI: 96.9%- 100%) and specificity of 97.1% (95%CI: 91.9%- 99.4%). The P. vivax specific B&S MG-LAMP had a sensitivity of 99.4% (96.6%- 100%) and specificity of 99.2% (95.5%- 100%) and the P. falciparum assay had a sensitivity of 100% (95%CI: 78.2%- 100%) and specificity of 99.3% (95%CI: 97.3%- 99.8%). The modified genus B&S MG-LAMP assay detected eight submicroscopic malaria cases (1.6%) which the species-specific assays did not identify. The turnaround time of B&S MG-LAMP was faster than expert microscopy with as many as 60 samples being processed per day by field technicians with limited training and utilizing a simple heat-block. The modified B&S MG-LAMP offers a simple and sensitive molecular test of choice for the detection of submicroscopic infections that can be used for mass screening in resources limited facilities in endemic settings nearing elimination and where a deployable test is required.

Field validation of a magneto-optical detection device (Gazelle) for portable point-of-care Plasmodium vivax diagnosis.

A major challenge for malaria is the lack of tools for accurate and timely diagnosis in the field which are critical for case management and surveillance. Microscopy along with rapid diagnostic tests are the current mainstay for malaria diagnosis in most endemic regions. However, these methods present several limitations. This study assessed the accuracy of Gazelle, a novel rapid malaria diagnostic device, from samples collected from the Peruvian Amazon between 2019 and 2020. Diagnostic accuracy was compared against microscopy and two rapid diagnostic tests (SD Bioline and BinaxNOW) using 18ssr nested-PCR as reference test. In addition, a real-time PCR assay (PET-PCR) was used for parasite quantification. Out of 217 febrile patients enrolled and tested, 180 specimens (85 P. vivax and 95 negatives) were included in the final analysis. Using nested-PCR as the gold standard, the sensitivity and specificity of Gazelle was 88.2% and 97.9%, respectively. Using a cutoff of 200 parasites/µl, Gazelle's sensitivity for samples with more than 200 p/uL was 98.67% (95%CI: 92.79% to 99.97%) whereas the sensitivity for samples lower than 200 p/uL (n = 10) was 12.5% (95%CI: 0.32% to 52.65%). Gazelle's sensitivity and specificity were statistically similar to microscopy (sensitivity = 91.8, specificity = 100%, p = 0.983) and higher than both SD Bioline (sensitivity = 82.4, specificity = 100%, p = 0.016) and BinaxNOW (sensitivity = 71.8%, specificity = 97.9%, p = 0.002). The diagnostic accuracy of Gazelle for malaria detection in P. vivax infections was comparable to light microscopy and superior to both RDTs even in the presence of low parasitemia infections. The performance of Gazelle makes it a valuable tool for malaria diagnosis and active case detection that can be utilized in different malaria-endemic regions.