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.

Evolutionary genetics of malaria.

Many standard-textbook population-genetic results apply to a wide range of species. Sometimes, however, population-genetic models and principles need to be tailored to a particular species. This is particularly true for malaria, which next to tuberculosis and HIV/AIDS ranks among the economically most relevant infectious diseases. Importantly, malaria is not one disease-five human-pathogenic species of Plasmodium exist. P. falciparum is not only the most severe form of human malaria, but it also causes the majority of infections. The second most relevant species, P. vivax, is already considered a neglected disease in several endemic areas. All human-pathogenic species have distinct characteristics that are not only crucial for control and eradication efforts, but also for the population-genetics of the disease. This is particularly true in the context of selection. Namely, fitness is determined by so-called fitness components, which are determined by the parasites live-history, which differs between malaria species. The presence of hypnozoites, i.e., dormant liver-stage parasites, which can cause disease relapses, is a distinct feature of P. vivax and P. ovale sp. In P. malariae inactivated blood-stage parasites can cause a recrudescence years after the infection was clinically cured. To properly describe population-genetic processes, such as the spread of anti-malarial drug resistance, these features must be accounted for appropriately. Here, we introduce and extend a population-genetic framework for the evolutionary dynamics of malaria, which applies to all human-pathogenic malaria species. The model focuses on, but is not limited to, the spread of drug resistance. The framework elucidates how the presence of dormant liver stage or inactivated blood stage parasites that act like seed banks delay evolutionary processes. It is shown that, contrary to standard population-genetic theory, the process of selection and recombination cannot be decoupled in malaria. Furthermore, we discuss the connection between haplotype frequencies, haplotype prevalence, transmission dynamics, and relapses or recrudescence in malaria.

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.

Molecular surveillance of the Plasmodium vivax multidrug resistance 1 gene in Peru between 2006 and 2015.

BACKGROUND: The high incidence of Plasmodium vivax infections associated with clinical severity and the emergence of chloroquine (CQ) resistance has posed a challenge to control efforts aimed at eliminating this disease. Despite conflicting evidence regarding the role of mutations of P. vivax multidrug resistance 1 gene (pvmdr1) in drug resistance, this gene can be a tool for molecular surveillance due to its variability and spatial patterns. METHODS: Blood samples were collected from studies conducted between 2006 and 2015 in the Northern and Southern Amazon Basin and the North Coast of Peru. Thick and thin blood smears were prepared for malaria diagnosis by microscopy and PCR was performed for detection of P. vivax monoinfections. The pvmdr1 gene was subsequently sequenced and the genetic data was used for haplotype and diversity analysis. RESULTS: A total of 550 positive P. vivax samples were sequenced; 445 from the Northern Amazon Basin, 48 from the Southern Amazon Basin and 57 from the North Coast. Eight non-synonymous mutations and three synonymous mutations were analysed in 4,395 bp of pvmdr1. Amino acid changes at positions 976F and 1076L were detected in the Northern Amazon Basin (12.8%) and the Southern Amazon Basin (4.2%) with fluctuations in the prevalence of both mutations in the Northern Amazon Basin during the course of the study that seemed to correspond with a malaria control programme implemented in the region. A total of 13 pvmdr1 haplotypes with non-synonymous mutations were estimated in Peru and an overall nucleotide diversity of π = 0.00054. The Northern Amazon Basin was the most diverse region (π = 0.00055) followed by the Southern Amazon and the North Coast (π = 0.00035 and π = 0.00014, respectively). CONCLUSION: This study showed a high variability in the frequencies of the 976F and 1076L polymorphisms in the Northern Amazon Basin between 2006 and 2015. The low and heterogeneous diversity of pvmdr1 found in this study underscores the need for additional research that can elucidate the role of this gene on P. vivax drug resistance as well as in vitro and clinical data that can clarify the extend of CQ resistance in Peru.

Comparison of real time and malachite-green based loop-mediated isothermal amplification assays for the detection of Plasmodium vivax and P. falciparum.

The current context of malaria elimination requires urgent development and implementation of highly sensitive and specific methods for prompt detection and treatment of malaria parasites. Such methods should overcome current delays in diagnosis, allow the detection of low-density infections and address the difficulties in accessing remote endemic communities. In this study, we assessed the performance of the RealAmp and malachite-green loop mediated isothermal amplification (MG-LAMP) methodologies, using microscopy and conventional nested-PCR as reference techniques. Both LAMP techniques were performed for Plasmodium genus, P. falciparum, and P. vivax identification using 136 whole blood samples collected from three communities located in the Peruvian Amazon basin. Turnaround time and costs of performing the LAMP assays were estimated and compared to that of microscopy and nested-PCR. Using nested-PCR as reference standard, we calculated the sensitivity, specificity and 95% confidence interval (CI) for all methods. RealAmp had a sensitivity of 92% (95% CI: 85-96.5%) and specificity of 100% (95% CI: 89.1-100%) for species detection; sensitivity and specificity of MG-LAMP were 94% (95% CI: 87.5-97.8%) and 100% (89.1-100%), respectively. Whereas microscopy showed 88.1% sensitivity (95% CI: 80.2-93.7%) and 100% specificity (95%: 89.1-100%). The turnaround time and costs of performing the LAMP assays were lower compared to those associated with nested-PCR but higher than those associated with microscopy. The two LAMP assays were shown to be more sensitive and simple to implement than microscopy. Both LAMP methodologies could be used as large-scale screening tests, but the MG-LAMP assay uses a simple, portable heat-block while the RealAmp requires a RealAmp machine or a real-time PCR machine. This makes the MG-LAMP an appropriate choice for malaria surveillance studies in endemic sites. Use of LAMP tests in active case detection of Plasmodium parasites could help to detect positive malaria cases early.

Molecular Epidemiology of Plasmodium falciparum Malaria Outbreak, Tumbes, Peru, 2010-2012.

During 2010-2012, an outbreak of 210 cases of malaria occurred in Tumbes, in the northern coast of Peru, where no Plasmodium falciparum malaria case had been reported since 2006. To identify the source of the parasite causing this outbreak, we conducted a molecular epidemiology investigation. Microsatellite typing showed an identical genotype in all 54 available isolates. This genotype was also identical to that of parasites isolated in 2010 in the Loreto region of the Peruvian Amazon and closely related to clonet B, a parasite lineage previously reported in the Amazon during 1998-2000. These findings are consistent with travel history of index case-patients. DNA sequencing revealed mutations in the Pfdhfr, Pfdhps, Pfcrt, and Pfmdr1 loci, which are strongly associated with resistance to chloroquine and sulfadoxine/pyrimethamine, and deletion of the Pfhrp2 gene. These results highlight the need for timely molecular epidemiology investigations to trace the parasite source during malaria reintroduction events.

Safety and comparability of controlled human Plasmodium falciparum infection by mosquito bite in malaria-naïve subjects at a new facility for sporozoite challenge.

BACKGROUND: Controlled human malaria infection (CHMI) studies which recapitulate mosquito-borne infection are a critical tool to identify protective vaccine and drug candidates for advancement to field trials. In partnership with the Walter Reed Army Institute of Research, the CHMI model was established at the Seattle Biomedical Research Institute's Malaria Clinical Trials Center (MCTC). Activities and reagents at both centers were aligned to ensure comparability and continued safety of the model. To demonstrate successful implementation, CHMI was performed in six healthy malaria-naïve volunteers. METHODS: All volunteers received NF54 strain Plasmodium falciparum by the bite of five infected Anopheles stephensi mosquitoes under controlled conditions and were monitored for signs and symptoms of malaria and for parasitemia by peripheral blood smear. Subjects were treated upon diagnosis with chloroquine by directly observed therapy. Immunological (T cell and antibody) and molecular diagnostic (real-time quantitative reverse transcriptase polymerase chain reaction [qRT-PCR]) assessments were also performed. RESULTS: All six volunteers developed patent parasitemia and clinical malaria. No serious adverse events occurred during the study period or for six months post-infection. The mean prepatent period was 11.2 days (range 9-14 days), and geometric mean parasitemia upon diagnosis was 10.8 parasites/µL (range 2-69) by microscopy. qRT-PCR detected parasites an average of 3.7 days (range 2-4 days) earlier than blood smears. All volunteers developed antibodies to the blood-stage antigen merozoite surface protein 1 (MSP-1), which persisted up to six months. Humoral and cellular responses to pre-erythrocytic antigens circumsporozoite protein (CSP) and liver-stage antigen 1 (LSA-1) were limited. CONCLUSION: The CHMI model was safe, well tolerated and characterized by consistent prepatent periods, pre-symptomatic diagnosis in 3/6 subjects and adverse event profiles as reported at established centers. The MCTC can now evaluate candidates in the increasingly diverse vaccine and drug pipeline using the CHMI model. TRIAL REGISTRATION: ClinicalTrials.gov NCT01058226.

Genetic variation and recurrent parasitaemia in Peruvian Plasmodium vivax populations.

BACKGROUND: Plasmodium vivax is a predominant species of malaria in parts of South America and there is increasing resistance to drugs to treat infections by P. vivax. The existence of latent hypnozoites further complicates the ability to classify recurrent infections as treatment failures due to relapse, recrudescence of hyponozoites or re-infections. Antigen loci are putatively under natural selection and may not be an optimal molecular marker to define parasite haplotypes in paired samples. Putatively neutral microsatellite loci, however, offer an assessment of neutral haplotypes. The objective here was to assess the utility of neutral microsatellite loci to reconcile cases of recurrent parasitaemia in Amazonian P. vivax populations in Peru. METHODS: Patient blood samples were collected from three locations in or around Iquitos in the Peruvian Amazon. Five putatively neutral microsatellite loci were characterized from 445 samples to ascertain the within and amongst population variation. A total of 30 day 0 and day of recurrent parasitaemia samples were characterized at microsatellite loci and five polymorphic antigen loci for haplotype classification. RESULTS: The genetic diversity at microsatellite loci was consistent with neutral levels of variation measured in other South American P. vivax populations. Results between antigen and microsatellite loci for the 30 day 0 and day of recurrent parasitaemia samples were the same for 80% of the pairs. The majority of non-concordant results were the result of differing alleles at microsatellite loci. This analysis estimates that 90% of the paired samples with the same microsatellite haplotype are unlikely to be due to a new infection. CONCLUSIONS: A population-level approach was used to yield a better estimate of the probability of a new infection versus relapse or recrudescence of homologous hypnozoites; hypnozoite activation was common for this cohort. Population studies are critical with the evaluation of genetic markers to assess P. vivax biology and epidemiology. The additional demonstration of microsatellite loci as neutral markers capable of distinguishing the origin of the recurrent parasites (new infection or originating from the patient) lends support to their use in assessment of treatment outcomes.

Failure of Supervised Chloroquine and Primaquine Regimen for the Treatment of Plasmodium vivax in the Peruvian Amazon.

The widespread use of primaquine (PQ) and chloroquine (CQ), together, may be responsible for the relatively few, isolated cases of chloroquine-resistant P. vivax (CQRPV) that have been reported from South America. We report here a case of P. vivax from the Amazon Basin of Peru that recurred against normally therapeutic blood levels of CQ. Four out of 540 patients treated with combination CQ and PQ had a symptomatic recurrence of P. vivax parasitemia within 35 days of treatment initiation, possibly indicating CQ failure. Whole blood total CQ level for one of these four subjects was 95 ng/ml on the day of recurrence. Based on published criteria that delineate CQRPV as a P. vivax parasitemia, either recrudescence or relapse, that appears against CQ blood levels >100 ng/mL, we document the occurrence of a P. vivax strain in Peru that had unusually high tolerance to the synergistic combination therapy of CQ + PQ that normally works quite well.

Prolonged protection provided by a single dose of atovaquone-proguanil for the chemoprophylaxis of Plasmodium falciparum malaria in a human challenge model.

BACKGROUND: We conducted a randomized, placebo-controlled, double-blind trial to establish the efficacy of atovaquone-proguanil to prevent malaria with the goal of simulating weekly dosing in a human Plasmodium falciparum challenge model. METHODS: Thirty volunteers randomly received 1 of the following dose regimens: (1) 250 milligrams of atovaquone and 100 milligrams of proguanil (250/100 milligrams) 1 day prior to infectious mosquito challenge (day -1), (2) 250/100 milligrams on day 4 after challenge, (3) 250/100 milligrams on day -7, (4) 500 milligrams of atovaquone and 200 milligrams of proguanil (500/200 milligrams) on day -7 or, (5) 1000 milligrams of atovaquone and 400 milligrams of proguanil (1000/400 milligrams) on day -7. All regimens included matching placebo such that all volunteers received identical pill numbers. Six volunteers served as open-label infectivity controls. Volunteers underwent mosquito sporozoite challenge with P. falciparum 3D7 strain. Follow-up consisted of serial microscopy and close clinical monitoring for 90 days. RESULTS: Six of 6 infectivity controls developed parasitemia as expected. Two of 5 evaluable volunteers receiving 250/100 milligrams 7 days prior to challenge and 1 of 6 volunteers receiving 1000/400 milligrams 7 days prior to challenge were microscopically diagnosed with malaria. All other volunteers were protected. Atovaquone exposure (area under the curve) during liver stage development was low in 2 of 3 volunteers with prophylactic failure (423 and 199 ng/mL × days compared with a mean for protected volunteers of 1903 ng/mL × days), as was peak concentration (165 and 81 ng/mL compared with a mean of 594 ng/mL in volunteers with prophylactic success). Elimination half-life was short in volunteers with prophylactic failure (2.4, 2.0, and 3.3 days compared with a mean of 4.1 days in volunteers with prophylactic success). CONCLUSIONS: Single-dose atovaquone-proguanil provides effective malaria chemoprophylaxis against P. falciparum challenge at dosing intervals supportive of weekly dosing. Postexposure prophylaxis 4 days after challenge was 100% effective.

Genome scanning of Amazonian Plasmodium falciparum shows subtelomeric instability and clindamycin-resistant parasites.

Here, we fully characterize the genomes of 14 Plasmodium falciparum patient isolates taken recently from the Iquitos region using genome scanning, a microarray-based technique that delineates the majority of single-base changes, indels, and copy number variants distinguishing the coding regions of two clones. We show that the parasite population in the Peruvian Amazon bears a limited number of genotypes and low recombination frequencies. Despite the essentially clonal nature of some isolates, we see high frequencies of mutations in subtelomeric highly variable genes and internal var genes, indicating mutations arising during self-mating or mitotic replication. The data also reveal that one or two meioses separate different isolates, showing that P. falciparum clones isolated from different individuals in defined geographical regions could be useful in linkage analyses or quantitative trait locus studies. Through pairwise comparisons of different isolates we discovered point mutations in the apicoplast genome that are close to known mutations that confer clindamycin resistance in other species, but which were hitherto unknown in malaria parasites. Subsequent drug sensitivity testing revealed over 100-fold increase of clindamycin EC(50) in strains harboring one of these mutations. This evidence of clindamycin-resistant parasites in the Amazon suggests that a shift should be made in health policy away from quinine + clindamycin therapy for malaria in pregnant women and infants, and that the development of new lincosamide antibiotics for malaria should be reconsidered.

Effects of point mutations in Plasmodium falciparum dihydrofolate reductase and dihydropterate synthase genes on clinical outcomes and in vitro susceptibility to sulfadoxine and pyrimethamine.

BACKGROUND: Sulfadoxine-pyrimethamine was a common first line drug therapy to treat uncomplicated falciparum malaria, but increasing therapeutic failures associated with the development of significant levels of resistance worldwide has prompted change to alternative treatment regimes in many national malaria control programs. METHODOLOGY AND FINDING: We conducted an in vivo therapeutic efficacy trial of sulfadoxine-pyrimethamine at two locations in the Peruvian Amazon enrolling 99 patients of which, 86 patients completed the protocol specified 28 day follow up. Our objective was to correlate the presence of polymorphisms in P. falciparum dihydrofolate reductase and dihydropteroate synthase to in vitro parasite susceptibility to sulfadoxine and pyrimethamine and to in vivo treatment outcomes. Inhibitory concentration 50 values of isolates increased with numbers of mutations (single [108N], sextuplet [BR/51I/108N/164L and 437G/581G]) and septuplet (BR/51I/108N/164L and 437G/540E/581G) with geometric means of 76 nM (35-166 nM), 582 nM (49-6890- nM) and 4909 (3575-6741 nM) nM for sulfadoxine and 33 nM (22-51 nM), 81 nM (19-345 nM), and 215 nM (176-262 nM) for pyrimethamine. A single mutation present in the isolate obtained at the time of enrollment from either dihydrofolate reductase (164L) or dihydropteroate synthase (540E) predicted treatment failure as well as any other single gene alone or in combination. Patients with the dihydrofolate reductase 164L mutation were 3.6 times as likely to be treatment failures [failures 85.4% (164L) vs 23.7% (I164); relative risk = 3.61; 95% CI: 2.14 - 6.64] while patients with the dihydropteroate synthase 540E were 2.6 times as likely to fail treatment (96.7% (540E) vs 37.5% (K540); relative risk = 2.58; 95% CI: 1.88 - 3.73). Patients with both dihydrofolate reductase 164L and dihydropteroate synthase 540E mutations were 4.1 times as likely to be treatment failures [96.7% vs 23.7%; RR = 4.08; 95% CI: 2.45 - 7.46] compared to patients having both wild forms (I164 and K540). CONCLUSIONS: In this part of the Amazon basin, it may be possible to predict treatment failure with sulfadoxine-pyrimethamine equally well by determination of either of the single mutations dihydrofolate reductase 164L or dihydropteroate synthase 540E. TRIAL REGISTRATION: ClinicalTrials.gov NCT00951106.

Dynamics of malaria drug resistance patterns in the Amazon basin region following changes in Peruvian national treatment policy for uncomplicated malaria.

Monitoring changes in the frequencies of drug-resistant and -sensitive genotypes can facilitate in vivo clinical trials to assess the efficacy of drugs before complete failure occurs. Peru changed its national treatment policy for uncomplicated malaria to artesunate (ART)-plus-mefloquine (MQ) combination therapy in the Amazon basin in 2001. We genotyped isolates collected in 1999 and isolates collected in 2006 to 2007 for mutations in the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes, multidrug resistance gene 1 (Pfmdr-1), the chloroquine (CQ) resistance transporter gene (Pfcrt), and the Ca(2+) ATPase gene (PfATP6); these have been shown to be involved in resistance to sulfadoxine-pyrimethamine (SP), MQ, CQ, and possibly ART, respectively. Microsatellite haplotypes around the Pfdhfr, Pfdhps, Pfcrt, and Pfmdr-1 loci were also determined. There was a significant decline in the highly SP resistant Pfdhfr and Pfdhps genotypes from 1999 to 2006. In contrast, a CQ-resistant Pfcrt genotype increased in frequency during the same period. Among five different Pfmdr-1 allelic forms noted in 1999, two genotypes increased in frequency while one genotype decreased by 2006. We also noted previously undescribed polymorphisms in the PfATP6 gene as well as an increase in the frequency of a deletion mutant during this period. In addition, microsatellite analysis revealed that the resistant Pfdhfr, Pfdhps, and Pfcrt genotypes have each evolved from a single founder haplotype, while Pfmdr-1 genotypes have evolved from at least two independent haplotypes. Importantly, this study demonstrates that the Peruvian triple mutant Pfdhps genotypes are very similar to those found in other parts of South America.

Unusual pattern of Plasmodium falciparum drug resistance in the northwestern Peruvian Amazon region.

High levels of Plasmodium falciparum resistance to both chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) have been documented throughout the Amazon Basin of South America. Because of reports about the persistent efficacy of both of these drugs in the northwestern Peruvian Amazon region, we carried out an evaluation of the therapeutic efficacy of chloroquine (25 mg/kg) and SP (25 mg/kg of the sulfadoxine component) for the treatment of uncomplicated P. falciparum infections at two sites: Ullpayacu and Pampa Hermoza/Alianza. A total of 111 patients were enrolled. Only 5 (14.3%) of the 35 patients who received CQ had an adequate clinical and parasitologic response (ACPR). Six subjects (17%) had early treatment failure, 1 (2.9%) had late clinical failure, and 23 (65.7%) had late parasitologic failure (LPF). Of the subjects treated with SP, 92.3% had ACPR and 7.7% had LPF. Based on these findings, it is clear that there are at least limited areas within the Peruvian Amazon region where P. falciparum strains continue to be sensitive to SP.