Influence of cytochrome P450 (CYP) 2C8 polymorphisms on the efficacy and tolerability of artesunate-amodiaquine treatment of uncomplicated Plasmodium falciparum malaria in Zanzibar.

BACKGROUND: The anti-malarial drug, amodiaquine, a commonly used, long-acting partner drug in artemisinin-based combination therapy, is metabolized to active desethyl-amodiaquine (DEAQ) by cytochrome P450 2C8 (CYP2C8). The CYP2C8 gene carries several polymorphisms including the more frequent minor alleles, CYP2C8*2 and CYP2C8*3. These minor alleles have been associated with decreased enzymatic activity, slowing the amodiaquine biotransformation towards DEAQ. This study aimed to assess the influence of these CYP2C8 polymorphisms on the efficacy and tolerability of artesunate-amodiaquine (AS-AQ) treatment for uncomplicated Plasmodium falciparum malaria in Zanzibar. METHODS: Dried blood spots on filter paper were collected from 618 children enrolled in two randomized clinical trials comparing AS-AQ and artemether-lumefantrine in 2002-2005 in Zanzibar. Study participant were under five years of age with uncomplicated falciparum malaria. Human CYP2C8*2 and CYP2C8*3 genotype frequencies were determined by PCR-restriction fragment length polymorphism. Statistical associations between CYP2C8*2 and/or CYP2C8*3 allele carriers and treatment outcome or occurrence of adverse events were assessed by Fisher's exact test. RESULTS: The allele frequencies of CYP2C8*2 and CYP2C8*3 were 17.5 % (95 % CI 15.4-19.7) and 2.7 % (95 % CI 1.8-3.7), respectively. There was no significant difference in the proportion of subjects carrying either CYP2C8*2 or CYP2C8*3 alleles amongst those with re-infections (44.1 %; 95 % CI 33.8-54.8) or those with recrudescent infections (48.3 %; 95 % CI 29.4-67.5), compared to those with an adequate clinical and parasitological response (36.7 %; 95 % CI 30.0-43.9) (P = 0.25 and P = 0.31, respectively). However, patients carrying either CYP2C8*2 or CYP2C8*3 alleles were significantly associated with an increased occurrence of non-serious adverse events, when compared with CYP2C8 *1/*1 wild type homozygotes (44.9 %; 95 % CI 36.1-54.0 vs. 28.1 %; 95 % CI 21.9-35.0, respectively; P = 0.003). CONCLUSIONS: CYP2C8 genotypes did not influence treatment efficacy directly, but the tolerability to AS-AQ may be reduced in subjects carrying the CYP2C8*2 and CYP2C8*3 alleles. The importance of this non-negligible association with regard to amodiaquine-based malaria chemotherapy warrants further investigation.

Increased Sensitivity of Plasmodium falciparum to Artesunate/Amodiaquine Despite 14 Years as First-Line Malaria Treatment, Zanzibar.

Artemisinin-based combination therapies (ACTs) are first-line treatments for uncomplicated Plasmodium falciparum malaria. ACT resistance is spreading in Asia but not yet in Africa. Reduced effects of ACT partner drugs have been reported but with little information regarding widely used artesunate/amodiaquine (ASAQ). We studied its efficacy in Zanzibar after 14 years as first-line treatment directly by an in vivo, single-armed trial and indirectly by prevalences of different genotypes in the P. falciparum chloroquine-resistance transporter, multidrug-resistance 1, and Kelch 13 propeller domain genes. In vivo efficacy was higher during 2017 (100%; 95% CI 97.4%-100%) than during 2002-2005 (94.7%; 95% CI 91.9%-96.7%) (p = 0.003). Molecular findings showed no artemisinin resistance-associated genotypes and major increases in genotypes associated with high sensitivity/efficacy for amodiaquine than before ASAQ was introduced. Thus, the efficacy of ASAQ is maintained and appears to be increased after long-term use in contrast to what is observed for other ACTs used in Africa.

Parasite clearance, cure rate, post-treatment prophylaxis and safety of standard 3-day versus an extended 6-day treatment of artemether-lumefantrine and a single low-dose primaquine for uncomplicated Plasmodium falciparum malaria in Bagamoyo district, Tanzania: a randomized controlled trial.

BACKGROUND: Artemisinin-based combination therapy (ACT) resistant Plasmodium falciparum represents an increasing threat to Africa. Extended ACT regimens from standard 3 to 6 days may represent a means to prevent its development and potential spread in Africa. METHODS: Standard 3-day treatment with artemether-lumefantrine (control) was compared to extended 6-day treatment and single low-dose primaquine (intervention); in a randomized controlled, parallel group, superiority clinical trial of patients aged 1-65 years with microscopy confirmed uncomplicated P. falciparum malaria, enrolled in Bagamoyo district, Tanzania. The study evaluated parasite clearance, including proportion of PCR detectable P. falciparum on days 5 and 7 (primary endpoint), cure rate, post-treatment prophylaxis, safety and tolerability. Clinical, and laboratory assessments, including ECG were conducted during 42 days of follow-up. Blood samples were collected for parasite detection (by microscopy and PCR), molecular genotyping and pharmacokinetic analyses. Kaplan-Meier survival analyses were done for both parasite clearance and recurrence. RESULTS: A total of 280 patients were enrolled, 141 and 139 in the control and intervention arm, respectively, of whom 121 completed 42 days follow-up in each arm. There was no difference in proportion of PCR positivity across the arms at day 5 (80/130 (61.5%) vs 89/134 (66.4%), p = 0.44), or day 7 (71/129 (55.0%) vs 70/134 (52.2%), p = 0.71). Day 42 microscopy determined cure rates (PCR adjusted) were 97.4% (100/103) and 98.3% (110/112), p = 0.65, in the control and intervention arm, respectively. Microscopy determined crude recurrent parasitaemia during follow-up was 21/121 (17.4%) in the control and 14/121 (11.6%) in the intervention arm, p = 0.20, and it took 34 days and 42 days in the respective arms for 90% of the patients to remain without recurrent parasitaemia. Lumefantrine exposure was significantly higher in intervention arm from D3 to D42, but cardiac, biochemical and haematological safety was high and similar in both arms. CONCLUSION: Extended 6-day artemether-lumefantrine treatment and a single low-dose of primaquine was not superior to standard 3-day treatment for ACT sensitive P. falciparum infections but, importantly, equally efficacious and safe. Thus, extended artemether-lumefantrine treatment may be considered as a future treatment regimen for ACT resistant P. falciparum, to prolong the therapeutic lifespan of ACT in Africa. Trial registration ClinicalTrials.gov, NCT03241901. Registered July 27, 2017 https://clinicaltrials.gov/show/NCT03241901.

Surveillance of genetic markers associated with Plasmodium falciparum resistance to artemisinin-based combination therapy in Pakistan, 2018-2019.

BACKGROUND: The spread of artemisinin resistance in the Greater Mekong Subregion of Southeast Asia poses a significant threat for current anti-malarial treatment guidelines globally. The aim of this study was to assess the current prevalence of molecular markers of drug resistance in Plasmodium falciparum in the four provinces with the highest malaria burden in Pakistan, after introducing artemether-lumefantrine as first-line treatment in 2017. METHODS: Samples were collected during routine malaria surveillance in Punjab, Sindh, Baluchistan, and Khyber Pakhtunkhwa provinces of Pakistan between January 2018 and February 2019. Plasmodium falciparum infections were confirmed by rapid diagnostic test or microscopy. Plasmodium falciparum positive isolates (n = 179) were screened by Sanger sequencing for single nucleotide polymorphisms (SNPs) in the P. falciparum kelch 13 (pfk13) propeller domain and in P. falciparum coronin (pfcoronin). SNPs in P. falciparum multidrug resistance 1 (pfmdr1) N86Y, Y184F, D1246Y and P. falciparum chloroquine resistance transporter (pfcrt) K76T were genotyped by PCR-restriction fragment length polymorphism. RESULTS: No artemisinin resistance associated SNPs were identified in the pfk13 propeller domain or in pfcoronin. The pfmdr1 N86, 184F, D1246 and pfcrt K76 alleles associated with reduced lumefantrine sensitivity were present in 83.8% (150/179), 16.9% (29/172), 100.0% (173/173), and 8.4% (15/179) of all infections, respectively. The chloroquine resistance associated pfcrt 76T allele was present in 98.3% (176/179) of infections. CONCLUSION: This study provides an update on the current prevalence of molecular markers associated with reduced P. falciparum sensitivity to artemether and/or lumefantrine in Pakistan, including a first baseline assessment of polymorphisms in pfcoronin. No mutations associated with artemisinin resistance were observed in pfk13 or pfcoronin. However, the prevalence of the pfmdr1 N86 and D1246 alleles, that have been associated with decreased susceptibility to lumefantrine, remain high. Although clinical and molecular data suggest that the current malaria treatment guidelines for P. falciparum are presently effective in Pakistan, close monitoring for artemisinin and lumefantrine resistance will be critical to ensure early detection and enhanced containment of emerging ACT resistance spreading across from Southeast Asia.

Falciparum malaria from coastal Tanzania and Zanzibar remains highly connected despite effective control efforts on the archipelago.

BACKGROUND: Tanzania's Zanzibar archipelago has made significant gains in malaria control over the last decade and is a target for malaria elimination. Despite consistent implementation of effective tools since 2002, elimination has not been achieved. Importation of parasites from outside of the archipelago is thought to be an important cause of malaria's persistence, but this paradigm has not been studied using modern genetic tools. METHODS: Whole-genome sequencing (WGS) was used to investigate the impact of importation, employing population genetic analyses of Plasmodium falciparum isolates from both the archipelago and mainland Tanzania. Ancestry, levels of genetic diversity and differentiation, patterns of relatedness, and patterns of selection between these two populations were assessed by leveraging recent advances in deconvolution of genomes from polyclonal malaria infections. RESULTS: Significant decreases in the effective population sizes were inferred in both populations that coincide with a period of decreasing malaria transmission in Tanzania. Identity by descent analysis showed that parasites in the two populations shared long segments of their genomes, on the order of 5 cM, suggesting shared ancestry within the last 10 generations. Even with limited sampling, two of isolates between the mainland and Zanzibar were identified that are related at the expected level of half-siblings, consistent with recent importation. CONCLUSIONS: These findings suggest that importation plays an important role for malaria incidence on Zanzibar and demonstrate the value of genomic approaches for identifying corridors of parasite movement to the island.

A cluster randomised controlled trial of two rounds of mass drug administration in Zanzibar, a malaria pre-elimination setting-high coverage and safety, but no significant impact on transmission.

BACKGROUND: Mass drug administration (MDA) has the potential to interrupt malaria transmission and has been suggested as a tool for malaria elimination in low-endemic settings. This study aimed to determine the effectiveness and safety of two rounds of MDA in Zanzibar, a pre-elimination setting. METHODS: A cluster randomised controlled trial was conducted in 16 areas considered as malaria hotspots, with an annual parasite index of > 0.8%. The areas were randomised to eight intervention and eight control clusters. The intervention included two rounds of MDA with dihydroartemisinin-piperaquine and single low-dose primaquine 4 weeks apart in May-June 2016. Primary and secondary outcomes were cumulative confirmed malaria case incidences 6 months post-MDA and parasite prevalences determined by PCR 3 months post-MDA. Additional outcomes included intervention coverage, treatment adherence, occurrence of adverse events, and cumulative incidences 3, 12, and 16 months post-MDA. RESULTS: Intervention coverage was 91.0% (9959/10944) and 87.7% (9355/10666) in the first and second rounds, respectively; self-reported adherence was 82.0% (881/1136) and 93.7% (985/1196). Adverse events were reported in 11.6% (147/1268) and 3.2% (37/1143) of post-MDA survey respondents after both rounds respectively. No serious adverse event was reported. No difference in cumulative malaria case incidence was observed between the control and intervention arms 6 months post-MDA (4.2 and 3.9 per 1000 population; p = 0.94). Neither was there a difference in PCR-determined parasite prevalences 3 months post-MDA (1.4% and 1.7%; OR = 1.0, p = 0.94), although having received at least the first MDA was associated with reduced odds of malaria infection (aOR = 0.35; p = 0.02). Among confirmed malaria cases at health facilities, 26.0% and 26.3% reported recent travel outside Zanzibar in the intervention and control shehias (aOR ≥ 85; p ≤ 0.001). CONCLUSIONS: MDA was implemented with high coverage, adherence, and tolerability. Despite this, no significant impact on transmission was observed. The findings suggest that two rounds of MDA in a single year may not be sufficient for a sustained impact on transmission in a pre-elimination setting, especially when the MDA impact is restricted by imported malaria. Importantly, this study adds to the limited evidence for the use of MDA in low transmission settings in sub-Saharan Africa. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02721186 (registration date: March 29, 2016).

Field deployment of loop-mediated isothermal amplification for centralized mass-screening of asymptomatic malaria in Zanzibar: a pre-elimination setting.

BACKGROUND: Molecular tools for detection of low-density asymptomatic Plasmodium infections are needed in malaria elimination efforts. This study reports results from the hitherto largest implementation of loop-mediated isothermal amplification (LAMP) for centralized mass screening of asymptomatic malaria in Zanzibar. METHODS: Healthy individuals present and willing to participate in randomly selected households in 60 villages throughout Zanzibar were screened for malaria by rapid diagnostic tests (RDT). In 50% of the study households, participants were asked to provide 60 µL of finger-prick blood for additional LAMP screening. LAMP was conducted in two centralized laboratories in Zanzibar, by trained technicians with limited or no previous experience of molecular methods. The LAMP assay was performed with Loopamp(TM) MALARIA Pan/Pf Detection Kit (Eiken Chemical Company, Japan). Samples positive for Plasmodium genus (Pan)-LAMP were re-tested using Plasmodium falciparum-specific LAMP kits. RESULTS: Paired RDT and LAMP samples were available from 3983 individuals. The prevalence of asymptomatic malaria was 0.5% (CI 95% 0.1-0.8) and 1.6% (CI 95% 1.1-2.2) by RDT and Pan-LAMP, respectively. LAMP detected 3.4 (CI 95% 2.2-5.2) times more Plasmodium positive samples than RDT. DNA contamination was experienced, but solved by repetitive decontamination of all equipment and reagents. CONCLUSIONS: LAMP is a simple and sensitive molecular tool, and has potential in active surveillance and mass-screening programmes for detection of low-density asymptomatic malaria in pre-elimination settings. However, in order to deploy LAMP more effectively in field settings, protocols may need to be adapted for processing larger numbers of samples. A higher throughput, affordable closed system would be ideal to avoid contamination.

Strong isolation by distance and evidence of population microstructure reflect ongoing Plasmodium falciparum transmission in Zanzibar.

The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania, and continued local transmission. To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo District on the coastal mainland from 2016-2018. Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias, suggests ongoing low level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Our data support importation as a main source of genetic diversity and contribution to the parasite population on Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive for malaria reemergence due to susceptible hosts and competent vectors.

Strong isolation by distance and evidence of population microstructure reflect ongoing Plasmodium falciparum transmission in Zanzibar.

Background: The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania and continued local transmission. Methods: To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo district on the coastal mainland from 2016 to 2018. Results: Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to the rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias, suggests ongoing low-level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Conclusions: Our data support importation as a main source of genetic diversity and contribution to the parasite population in Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive to malaria reemergence due to susceptible hosts and competent vectors. Funding: This research was funded by the National Institutes of Health, grants R01AI121558, R01AI137395, R01AI155730, F30AI143172, and K24AI134990. Funding was also contributed from the Swedish Research Council, Erling-Persson Family Foundation, and the Yang Fund. RV acknowledges funding from the MRC Centre for Global Infectious Disease Analysis (reference MR/R015600/1), jointly funded by the UK Medical Research Council (MRC) and the UK Foreign, Commonwealth & Development Office (FCDO), under the MRC/FCDO Concordat agreement and is also part of the EDCTP2 program supported by the European Union. RV also acknowledges funding by Community Jameel.

Usefulness of Plasmodium falciparum-specific rapid diagnostic tests for assessment of parasite clearance and detection of recurrent infections after artemisinin-based combination therapy.

BACKGROUND: Rapid diagnostic test (RDT) is an important tool for parasite-based malaria diagnosis. High specificity of RDTs to distinguish an active Plasmodium falciparum infection from residual antigens from a previous infection is crucial in endemic areas where residents are repeatedly exposed to malaria. The efficiency of two RDTs based on histidine-rich protein 2 (HRP2) and lactate dehydrogenase (LDH) antigens were studied and compared with two microscopy techniques (Giemsa and acridine orange-stained blood smears) and real-time polymerase chain reaction (PCR) for assessment of initial clearance and detection of recurrent P. falciparum infections after artemisinin-based combination therapy (ACT) in a moderately high endemic area of rural Tanzania. METHODS: In this exploratory study 53 children < five years with uncomplicated P. falciparum malaria infection were followed up on nine occasions, i.e., day 1, 2, 3, 7, 14, 21, 28, 35 and 42, after initiation of artemether-lumefantrine treatment. At each visit capillary blood samples was collected for the HRP2 and LDH-based RDTs, Giemsa and acridine orange-stained blood smears for microscopy and real-time PCR. Assessment of clearance times and detection of recurrent P. falciparum infections were done for all diagnostic methods. RESULTS: The median clearance times were 28 (range seven to >42) and seven (two to 14) days for HRP2 and LDH-based RDTs, two (one to seven) and two (one to 14) days for Giemsa and acridine orange-stained blood smear and two (one to 28) days for real-time PCR. RDT specificity against Giemsa-stained blood smear microscopy was 21% for HRP2 on day 14, reaching 87% on day 42, and ≥96% from day 14 to 42 for LDH. There was no significant correlation between parasite density at enrolment and duration of HRP2 positivity (r = 0.13, p = 0.34). Recurrent malaria infections occurred in ten (19%) children. The HRP2 and LDH-based RDTs did not detect eight and two of the recurrent infections, respectively. CONCLUSION: The LDH-based RDT was superior to HRP2-based for monitoring of treatment outcome and detection of recurrent infections after ACT in this moderately high transmission setting. The results may have implications for the choice of RDT devices in similar transmission settings for improved malaria case management. TRIAL REGISTRATION: Clinicaltrials.gov, NCT01843764.

Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability.

BACKGROUND: The need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations. METHODS: DNA was extracted from two RDT devices (Paracheck-Pf® and SD Bioline Malaria Pf/Pan®), seeded in vitro with 10-fold dilutions of cultured 3D7 P. falciparum parasites diluted in malaria negative whole blood. The level of P. falciparum detection was determined for each extraction method and RDT device with multiple nested-PCR and real-time PCR assays. The field applicability was tested on 855 paired RDT (Paracheck-Pf) and filter paper (Whatman® 3MM) blood samples (734 RDT negative and 121 RDT positive samples) collected from febrile patients in Zanzibar 2010. RDT positive samples were genotyped at four key single nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt as well as for pfmdr1 copy number, all associated with anti-malarial drug resistance. RESULTS: The P. falciparum DNA detection limit varied with RDT device and extraction method. Chelex-100 extraction performed best for all extraction matrixes. There was no statistically significant difference in PCR detection rates in DNA extracted from RDTs and filter paper field samples. Similarly there were no significant differences in the PCR success rates and genotyping outcomes for the respective SNPs in the 121 RDT positive samples. CONCLUSIONS: The results support RDTs as a valuable source of parasite DNA and provide evidence for RDT-DNA extraction for improved malaria case detection, molecular drug resistance surveillance, and RDT quality control.

Decreased prevalence of Plasmodium falciparum resistance markers to amodiaquine despite its wide scale use as ACT partner drug in Zanzibar.

BACKGROUND: Zanzibar has recently undergone a rapid decline in Plasmodium falciparum transmission following combined malaria control interventions with artemisinin-based combination therapy (ACT) and integrated vector control. Artesunate-amodiaquine (ASAQ) was implemented as first-line treatment for uncomplicated P. falciparum malaria in Zanzibar in 2003. Resistance to amodiaquine has been associated with the single nucleotide polymorphism (SNP) alleles pfcrt 76T, pfmdr1 86Y, 184Y and 1246Y. An accumulation of these SNP alleles in the parasite population over time might threaten ASAQ efficacy.The aim of this study was to assess whether prolonged use of ASAQ as first-line anti-malarial treatment selects for P. falciparum SNPs associated with resistance to the ACT partner drug amodiaquine. METHODS: The individual as well as the combined SNP allele prevalence were compared in pre-treatment blood samples from patients with uncomplicated P. falciparum malaria enrolled in clinical trials conducted just prior to the introduction of ASAQ in 2002-2003 (n = 208) and seven years after wide scale use of ASAQ in 2010 (n = 122). RESULTS: There was a statistically significant decrease of pfcrt 76T (96-63%), pfmdr1 86Y (75-52%), 184Y (83-72%), 1246Y (28-16%) and the most common haplotypes pfcrt/pfmdr1 TYYD (46-26%) and TYYY (17-8%), while an increase of pfcrt/pfmdr1 KNFD (0.4-14%) and KNYD (1-12%). CONCLUSIONS: This is the first observation of a decreased prevalence of pfcrt 76T, pfmdr1 86Y, 184Y and 1246Y in an African setting after several years of extensive ASAQ use as first-line treatment for uncomplicated malaria. This may support sustained efficacy of ASAQ on Zanzibar, although it was unexpected considering that all these SNPs have previously been associated with amodiaquine resistance. The underlying factors of these results are unclear. Genetic dilution by imported P. falciparum parasites from mainland Tanzania, a de-selection by artesunate per se and/or an associated fitness cost might represent contributing factors. More detailed studies on temporal trends of molecular markers associated with amodiaquine resistance are required to improve the understanding of this observation.

An update on pharmacogenetic factors influencing the metabolism and toxicity of artemisinin-based combination therapy in the treatment of malaria.

INTRODUCTION: Artemisinin-based combination therapies (ACTs) are recommended first-line antimalarials for uncomplicated Plasmodium falciparum malaria. Pharmacokinetic/pharmacodynamic variation associated with ACT drugs and their effect is documented. It is accepted to an extent that inter-individual variation is genetically driven, and should be explored for optimized antimalarial use. AREAS COVERED: We provide an update on the pharmacogenetics of ACT antimalarial disposition. Beyond presently used antimalarials, we also refer to information available for the most notable next-generation drugs under development. The bibliographic approach was based on multiple Boolean searches on PubMed covering all recent publications since our previous review. EXPERT OPINION: The last 10 years have witnessed an increase in our knowledge of ACT pharmacogenetics, including the first clear examples of its contribution as an exacerbating factor for drug-drug interactions. This knowledge gap is still large and is likely to widen as a new wave of antimalarial drug is looming, with few studies addressing their pharmacogenetics. Clinically useful pharmacogenetic markers are still not available, in particular, from an individual precision medicine perspective. A better understanding of the genetic makeup of target populations can be valuable for aiding decisions on mass drug administration implementation concerning region-specific antimalarial drug and dosage options.

Performance and Application of Commercially Available Loop-Mediated Isothermal Amplification (LAMP) Kits in Malaria Endemic and Non-Endemic Settings.

Loop-mediated isothermal amplification (LAMP) is a sensitive molecular tool suitable for use as a near point-of-care test for the diagnosis of malaria. Recent meta-analyses have detailed high sensitivity and specificity of malaria LAMP when compared to microscopy, rapid diagnostic tests, and polymerase chain reaction in both endemic and non-endemic settings. Despite this, the use of malaria LAMP has primarily been limited to research settings to date. In this review, we aim to assess to what extent commercially available malaria LAMP kits have been applied in different settings, and to identify possible obstacles that may have hindered their use from being adopted further. In order to address this, we conducted a literature search in PubMed.gov using the search terms (((LAMP) OR (Loop-mediated isothermal amplification)) AND ((Malaria) OR (Plasmodium))). Focusing primarily on studies employing one of the commercially available kits, we then selected three key areas of LAMP application for further review: the performance and application of LAMP in malaria endemic settings including low transmission areas; LAMP for malaria screening during pregnancy; and malaria LAMP in returning travelers in non-endemic settings.

Why Asymptomatic Plasmodium falciparum Infections Are Common in Low-Transmission Settings.

Plasmodium falciparum infections in low-transmission settings are often asymptomatic with low parasite densities despite low herd immunity. Based on studies in Zanzibar, this may be due to parasitic (nonvirulence) rather than host (immunity) factors. In high-transmission settings, high replication rate and virulence represents a competitive advantage, whereas in low-transmission settings nonvirulent parasites escape both competition and treatment. Such parasites also survive longer in low-transmission settings due to lower host immunity response and less frequent indirect drug exposure. This has major implications for optimal malaria control and elimination strategies.

Detection of Plasmodium falciparum by Light Microscopy, Loop-Mediated Isothermal Amplification, and Polymerase Chain Reaction on Day 3 after Initiation of Artemether-Lumefantrine Treatment for Uncomplicated Malaria in Bagamoyo District, Tanzania: A Comparative Trial.

Microscopy-determined Plasmodium falciparum positivity rates exceeding 10% on day 3 after initiation of artemisinin-based combination therapy (ACT) is an important indicator of artemisinin resistance. However, microscopy does not detect low-density parasitemia, contrary to molecular tools such as loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). We compared microscopy, LAMP, and PCR for detection of P. falciparum on day 3 after ACT in 256 patients with uncomplicated malaria in Bagamoyo District, Tanzania. Day 3 positivity rates were 0%, 84.8%, and 84.4% for each method, respectively. The sensitivity and specificity of LAMP against PCR was 100% (95% CI, 96.1-100) and 77.4% (95% CI, 58.9-90.4) when quantitative PCR-determined parasite densities were ≥ two parasites/µL. Loop-mediated isothermal amplification had comparable diagnostic accuracy to PCR and could potentially represent a field-friendly tool for determining day 3 positivity rates. However, what day 3 P. falciparum positivity determined using molecular methods represents needs to be further elucidated.

The temporal dynamics and infectiousness of subpatent Plasmodium falciparum infections in relation to parasite density.

Malaria infections occurring below the limit of detection of standard diagnostics are common in all endemic settings. However, key questions remain surrounding their contribution to sustaining transmission and whether they need to be detected and targeted to achieve malaria elimination. In this study we analyse a range of malaria datasets to quantify the density, detectability, course of infection and infectiousness of subpatent infections. Asymptomatically infected individuals have lower parasite densities on average in low transmission settings compared to individuals in higher transmission settings. In cohort studies, subpatent infections are found to be predictive of future periods of patent infection and in membrane feeding studies, individuals infected with subpatent asexual parasite densities are found to be approximately a third as infectious to mosquitoes as individuals with patent (asexual parasite) infection. These results indicate that subpatent infections contribute to the infectious reservoir, may be long lasting, and require more sensitive diagnostics to detect them in lower transmission settings.

Author Correction: The temporal dynamics and infectiousness of subpatent Plasmodium falciparum infections in relation to parasite density.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Absence of Plasmodium falciparum K13 Propeller Domain Polymorphisms among Field Isolates Collected from the Brazilian Amazon Basin between 1984 and 2011.

Artemisinin resistance, presently confined to Southeast Asia and associated with mutations in the Plasmodium falciparum K13 (PfK13) propeller domain, represents a serious threat to global malaria control. This study aimed to provide baseline information for future artemisinin resistance surveillance, by analyzing the PfK13 propeller domain in P. falciparum field isolates collected from the Brazilian Amazon Basin between 1984 and 2011. A total of 152 P. falciparum mono-infections were assessed, of which 118 (78%) were collected before and 34 (22%) after the introduction of artemisinin-based combination therapy (ACT) in 2006. An 849-base pair fragment encoding the PfK13 propeller was amplified by nested polymerase chain reaction and sequenced in both directions. The sequences were compared with the reference sequence of P. falciparum 3D7. All samples showed wild-type sequences, thus, no mutations were observed. The results are in agreement with other recent reports and do not provide evidence for presence of PfK13 propeller domain polymorphisms associated with artemisinin resistance among P. falciparum field isolates in the Brazilian Amazon Basin neither before nor after the implementation of ACT.

Field Evaluation of a High Throughput Loop Mediated Isothermal Amplification Test for the Detection of Asymptomatic Plasmodium Infections in Zanzibar.

BACKGROUND: New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. METHODS: HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a community-based survey in 2015. RESULTS: The PCR and HTP-LAMP determined malaria prevalences were 1.6% (95%CI 1.3-2.4) and 0.7% (95%CI 0.4-1.1), respectively. The sensitivity of HTP-LAMP compared to PCR was 40.8% (CI95% 27.0-55.8) and the specificity was 99.9% (CI95% 99.8-100). For the PCR positive samples, there was no statistically significant difference between the geometric mean parasite densities among the HTP-LAMP positive (2.5 p/µL, range 0.2-770) and HTP-LAMP negative (1.4 p/µL, range 0.1-7) samples (p = 0.088). Two lab technicians analysed up to 282 samples per day and the HTP-LAMP method was experienced as user friendly. CONCLUSIONS: Although field applicable, this high throughput format of LAMP as used here was not sensitive enough to be recommended for detection of asymptomatic low-density infections in areas like Zanzibar, approaching malaria elimination.