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

BACKGROUND: Therapeutic efficacy studies (TESs) and detection of molecular markers of drug resistance are recommended by the World Health Organization (WHO) to monitor the efficacy of artemisinin-based combination therapy (ACT). This study assessed the trends of molecular markers of artemisinin resistance and/or reduced susceptibility to lumefantrine using samples collected in TES conducted in Mainland Tanzania from 2016 to 2021. METHODS: A 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). RESULTS: Sequencing 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 the WHO as molecular markers of resistance. 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%. CONCLUSION: This 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 artemisinin partial resistance 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 ACT.

Molecular surveillance for anti-malarial drug resistance and genetic diversity of Plasmodium falciparum after chloroquine and sulfadoxine-pyrimethamine withdrawal in Quibdo, Colombia, 2018.

BACKGROUND: Resistance to anti-malarial drugs is associated with polymorphisms in target genes and surveillance for these molecular markers is important to detect the emergence of mutations associated with drug resistance and signal recovering sensitivity to anti-malarials previously used. METHODS: The presence of polymorphisms in genes associated with Plasmodium falciparum resistance to chloroquine and sulfadoxine-pyrimethamine was evaluated by Sanger sequencing, in 85 P. falciparum day of enrollment samples from a therapeutic efficacy study of artemether-lumefantrine conducted in 2018-2019 in Quibdo, Colombia. Samples were genotyped to assess mutations in pfcrt (codons 72-76), pfdhfr (codons 51, 59, 108, and 164), and pfdhps genes (codons 436, 437, 540, and 581). Further, the genetic diversity of infections using seven neutral microsatellites (NMSs) (C2M34, C3M69, Poly α, TA1, TA109, 2490, and PfPK2) was assessed. RESULTS: All isolates carried mutant alleles for pfcrt (K76T and N75E), and for pfdhfr (N51I and S108N), while for pfdhps, mutations were observed only for codon A437G (32/73, 43.8%). Fifty samples (58.8%) showed a complete neutral microsatellites (NMS) profile. The low mean number of alleles (2 ± 0.57) per locus and mean expected heterozygosity (0.17 ± 0.03) showed a reduced genetic diversity. NMS multilocus genotypes (MMG) were built and nine MMG were identified. CONCLUSIONS: Overall, these findings confirm the fixation of chloroquine and pyrimethamine-resistant alleles already described in the literature, implying that these drugs are not currently appropriate for use in Colombia. In contrast, mutations in the pfdhps gene were only observed at codon 437, an indication that full resistance to sulfadoxine has not been achieved in Choco. MMGs found matched the clonal lineage E variant 1 previously reported in northwestern Colombia.

Plasmodium falciparum kelch 13 Mutations, 9 Countries in Africa, 2014-2018.

The spread of drug resistance to antimalarial treatments poses a serious public health risk globally. To combat this risk, molecular surveillance of drug resistance is imperative. We report the prevalence of mutations in the Plasmodium falciparum kelch 13 propeller domain associated with partial artemisinin resistance, which we determined by using Sanger sequencing samples from patients enrolled in therapeutic efficacy studies from 9 sub-Saharan countries during 2014-2018. Of the 2,865 samples successfully sequenced before treatment (day of enrollment) and on the day of treatment failure, 29 (1.0%) samples contained 11 unique nonsynonymous mutations and 83 (2.9%) samples contained 27 unique synonymous mutations. Two samples from Kenya contained the S522C mutation, which has been associated with delayed parasite clearance; however, no samples contained validated or candidate artemisinin-resistance mutations.

Low prevalence of highly sulfadoxine-resistant dihydropteroate synthase alleles in Plasmodium falciparum isolates in Benin.

BACKGROUND: In 2004, in response to high levels of treatment failure associated with sulfadoxine-pyrimethamine (SP) resistance, Benin changed its first-line malaria treatment from SP to artemisinin-based combination therapy for treatment of uncomplicated Plasmodium falciparum malaria. Resistance to SP is conferred by accumulation of single nucleotide polymorphisms (SNPs) in P. falciparum genes involved in folate metabolism, dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps), targeted by pyrimethamine and sulfadoxine, respectively. Because SP is still used for intermittent preventive treatment in pregnant women (IPTp) and seasonal malaria chemoprevention (SMCP) in Benin, the prevalence of Pfdhfr and Pfdhps SNPs in P. falciparum isolates collected in 2017 were investigated. METHODS: This study was carried out in two sites where the transmission of P. falciparum malaria is hyper-endemic: Klouékanmey and Djougou. Blood samples were collected from 178 febrile children 6-59 months old with confirmed uncomplicated P. falciparum malaria and were genotyped for SNPs associated with SP resistance. RESULTS: The Pfdhfr triple mutant IRN (N51I, C59R, and S108N) was the most prevalent (84.6%) haplotype and was commonly found with the Pfdhps single mutant A437G (50.5%) or with the Pfdhps double mutant S436A and A437G (33.7%). The quintuple mutant, Pfdhfr IRN/Pfdhps GE (A437G and K540E), was rarely observed (0.8%). The A581G and A613S mutant alleles were found in 2.6 and 3.9% of isolates, respectively. Six isolates (3.9%) were shown to harbour a mutation at codon I431V, recently identified in West African parasites. CONCLUSIONS: This study showed that Pfdhfr triple IRN mutants are near fixation in this population and that the highly sulfadoxine-resistant Pfdhps alleles are not widespread in Benin. These data support the continued use of SP for chemoprevention in these study sites, which should be complemented by periodic nationwide molecular surveillance to detect emergence of resistant genotypes.

Molecular surveillance for polymorphisms associated with artemisinin-based combination therapy resistance in Plasmodium falciparum isolates collected in Mozambique, 2018.

BACKGROUND: Due to the threat of emerging anti-malarial resistance, the World Health Organization recommends incorporating surveillance for molecular markers of anti-malarial resistance into routine therapeutic efficacy studies (TESs). In 2018, a TES of artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) was conducted in Mozambique, and the prevalence of polymorphisms in the pfk13, pfcrt, and pfmdr1 genes associated with drug resistance was investigated. METHODS: Children aged 6-59 months were enrolled in four study sites. Blood was collected and dried on filter paper from participants who developed fever within 28 days of initial malaria treatment. All samples were first screened for Plasmodium falciparum using a multiplex real-time PCR assay, and polymorphisms in the pfk13, pfcrt, and pfmdr1 genes were investigated by Sanger sequencing. RESULTS: No pfk13 mutations, associated with artemisinin partial resistance, were observed. The only pfcrt haplotype observed was the wild type CVMNK (codons 72-76), associated with chloroquine sensitivity. Polymorphisms in pfmdr1 were only observed at codon 184, with the mutant 184F in 43/109 (39.4%) of the samples, wild type Y184 in 42/109 (38.5%), and mixed 184F/Y in 24/109 (22.0%). All samples possessed N86 and D1246 at these two codons. CONCLUSION: In 2018, no markers of artemisinin resistance were documented. Molecular surveillance should continue to monitor the prevalence of these markers to inform decisions on malaria treatment in Mozambique.

Efficacy of artesunate-amodiaquine and artemether-lumefantrine for uncomplicated Plasmodium falciparum malaria in Madagascar, 2018.

BACKGROUND: Since 2005, artemisinin-based combination therapy (ACT) has been recommended to treat uncomplicated falciparum malaria in Madagascar. Artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) are the first- and second-line treatments, respectively. A therapeutic efficacy study was conducted to assess ACT efficacy and molecular markers of anti-malarial resistance. METHODS: Children aged six months to 14 years with uncomplicated falciparum malaria and a parasitaemia of 1000-100,000 parasites/µl determined by microscopy were enrolled from May-September 2018 in a 28-day in vivo trial using the 2009 World Health Organization protocol for monitoring anti-malarial efficacy. Participants from two communes, Ankazomborona (tropical, northwest) and Matanga (equatorial, southeast), were randomly assigned to ASAQ or AL arms at their respective sites. PCR correction was achieved by genotyping seven neutral microsatellites in paired pre- and post-treatment samples. Genotyping assays for molecular markers of resistance in the pfk13, pfcrt and pfmdr1 genes were conducted. RESULTS: Of 344 patients enrolled, 167/172 (97%) receiving ASAQ and 168/172 (98%) receiving AL completed the study. For ASAQ, the day-28 cumulative PCR-uncorrected efficacy was 100% (95% CI 100-100) and 95% (95% CI 91-100) for Ankazomborona and Matanga, respectively; for AL, it was 99% (95% CI 97-100) in Ankazomborona and 83% (95% CI 76-92) in Matanga. The day-28 cumulative PCR-corrected efficacy for ASAQ was 100% (95% CI 100-100) and 98% (95% CI 95-100) for Ankazomborona and Matanga, respectively; for AL, it was 100% (95% CI 99-100) in Ankazomborona and 95% (95% CI 91-100) in Matanga. Of 83 successfully sequenced samples for pfk13, no mutation associated with artemisinin resistance was observed. A majority of successfully sequenced samples for pfmdr1 carried either the NFD or NYD haplotypes corresponding to codons 86, 184 and 1246. Of 82 successfully sequenced samples for pfcrt, all were wild type at codons 72-76. CONCLUSION: PCR-corrected analysis indicated that ASAQ and AL have therapeutic efficacies above the 90% WHO acceptable cut-off. No genetic evidence of resistance to artemisinin was observed, which is consistent with the clinical outcome data. However, the most common pfmdr1 haplotypes were NYD and NFD, previously associated with tolerance to lumefantrine.

Efficacy and safety of artemether-lumefantrine and dihydroartemisinin-piperaquine for the treatment of uncomplicated Plasmodium falciparum malaria and prevalence of molecular markers associated with artemisinin and partner drug resistance in Uganda.

BACKGROUND: In Uganda, artemether-lumefantrine (AL) is first-line therapy and dihydroartemisinin-piperaquine (DP) second-line therapy for the treatment of uncomplicated malaria. This study evaluated the efficacy and safety of AL and DP in the management of uncomplicated falciparum malaria and measured the prevalence of molecular markers of resistance in three sentinel sites in Uganda from 2018 to 2019. METHODS: This was a randomized, open-label, phase IV clinical trial. Children aged 6 months to 10 years with uncomplicated falciparum malaria were randomly assigned to treatment with AL or DP and followed for 28 and 42 days, respectively. Genotyping was used to distinguish recrudescence from new infection, and a Bayesian algorithm was used to assign each treatment failure a posterior probability of recrudescence. For monitoring resistance, Pfk13 and Pfmdr1 genes were Sanger sequenced and plasmepsin-2 copy number was assessed by qPCR. RESULTS: There were no early treatment failures. The uncorrected 28-day cumulative efficacy of AL ranged from 41.2 to 71.2% and the PCR-corrected cumulative 28-day efficacy of AL ranged from 87.2 to 94.4%. The uncorrected 28-day cumulative efficacy of DP ranged from 95.8 to 97.9% and the PCR-corrected cumulative 28-day efficacy of DP ranged from 98.9 to 100%. The uncorrected 42-day efficacy of DP ranged from 73.5 to 87.4% and the PCR-corrected 42-day efficacy of DP ranged from 92.1 to 97.5%. There were no reported serious adverse events associated with any of the regimens. No resistance-associated mutations in the Pfk13 gene were found in the successfully sequenced samples. In the AL arm, the NFD haplotype (N86Y, Y184F, D1246Y) was the predominant Pfmdr1 haplotype, present in 78 of 127 (61%) and 76 of 110 (69%) of the day 0 and day of failure samples, respectively. All the day 0 samples in the DP arm had one copy of the plasmepsin-2 gene. CONCLUSIONS: DP remains highly effective and safe for the treatment of uncomplicated malaria in Uganda. Recurrent infections with AL were common. In Busia and Arua, the 95% confidence interval for PCR-corrected AL efficacy fell below 90%. Further efficacy monitoring for AL, including pharmacokinetic studies, is recommended. Trial registration The trail was also registered with the ISRCTN registry with study Trial No. PACTR201811640750761.

Therapeutic efficacy of artemether-lumefantrine and artesunate-amodiaquine for the treatment of uncomplicated Plasmodium falciparum malaria in Mali, 2015-2016.

BACKGROUND: The current first-line treatments for uncomplicated malaria recommended by the National Malaria Control Programme in Mali are artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ). From 2015 to 2016, an in vivo study was carried out to assess the clinical and parasitological responses to AL and ASAQ in Sélingué, Mali. METHODS: Children between 6 and 59 months of age with uncomplicated Plasmodium falciparum infection and 2000-200,000 asexual parasites/µL of blood were enrolled, randomly assigned to either AL or ASAQ, and followed up for 42 days. Uncorrected and PCR-corrected efficacy results at days 28 and 42. were calculated. Known markers of resistance in the Pfk13, Pfmdr1, and Pfcrt genes were assessed using Sanger sequencing. RESULTS: A total of 449 patients were enrolled: 225 in the AL group and 224 in the ASAQ group. Uncorrected efficacy at day 28 was 83.4% (95% CI 78.5-88.4%) in the AL arm and 93.1% (95% CI 89.7-96.5%) in the ASAQ arm. The per protocol PCR-corrected efficacy at day 28 was 91.0% (86.0-95.9%) in the AL arm and 97.1% (93.6-100%) in the ASAQ arm. ASAQ was significantly (p < 0.05) better than AL for each of the aforementioned efficacy outcomes. No mutations associated with artemisinin resistance were identified in the Pfk13 gene. Overall, for Pfmdr1, the N86 allele and the NFD haplotype were the most common. The NFD haplotype was significantly more prevalent in the post-treatment than in the pre-treatment isolates in the AL arm (p < 0.01) but not in the ASAQ arm. For Pfcrt, the CVIET haplotype was the most common. CONCLUSIONS: The findings indicate that both AL and ASAQ remain effective for the treatment of uncomplicated malaria in Sélingué, Mali.

The endoplasmic reticulum 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 uncharacterised ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localises 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 eIF2α kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway.

Molecular and epidemiological characterization of imported malaria cases in Chile.

BACKGROUND: Chile is one of the South American countries certified as malaria-free since 1945. However, the recent increase of imported malaria cases and the presence of the vector Anopheles pseudopunctipennis in previously endemic areas in Chile require an active malaria surveillance programme. METHODS: Specimens from 268 suspected malaria cases-all imported-collected between 2015 and 2018 at the Public Health Institute of Chile (ISP), were diagnosed by microscopy and positive cases were included for epidemiological analysis. A photo-induced electron transfer fluorogenic primer real-time PCR (PET-PCR) was used to confirm the presence of malaria parasites in available blood samples. Sanger sequencing of drug resistance molecular markers (pfk13, pfcrt and pfmdr1) and microsatellite (MS) analysis were performed in confirmed Plasmodium falciparum samples and results were related to origin of infection. RESULTS: Out of the 268 suspected cases, 65 were Plasmodium spp. positive by microscopy. A total of 63% of the malaria patients were male and 37% were female; 43/65 of the patients acquired infections in South American endemic countries. Species confirmation of available blood samples by PET-PCR revealed that 15 samples were positive for P. falciparum, 27 for Plasmodium vivax and 4 were mixed infections. The P. falciparum samples sequenced contained four mutant pfcrt genotypes (CVMNT, CVMET, CVIET and SVMNT) and three mutant pfmdr1 genotypes (Y184F/S1034C/N1042D/D1246Y, Y184F/N1042D/D1246Y and Y184F). MS analysis confirmed that all P. falciparum samples presented different haplotypes according to the suspected country of origin. Four patients with P. vivax infection returned to the health facilities due to relapses. CONCLUSION: The timely detection of polymorphisms associated with drug resistance will contribute to understanding if current drug policies in the country are appropriate for treatment of imported malaria cases and provide information about the most frequent resistant genotypes entering Chile.

WHO malaria nucleic acid amplification test external quality assessment scheme: results of distribution programmes one to three.

BACKGROUND: The World Health Organization (WHO) recommends parasite-based diagnosis of malaria. In recent years, there has been surge in the use of various kinds of nucleic-acid amplification based tests (NAATs) for detection and identification of Plasmodium spp. to support clinical care in high-resource settings and clinical and epidemiological research worldwide. However, these tests are not without challenges, including lack (or limited use) of standards and lack of reproducibility, due in part to variation in protocols amongst laboratories. Therefore, there is a need for rigorous quality control, including a robust external quality assessment (EQA) scheme targeted towards malaria NAATs. To this effect, the WHO Global Malaria Programme worked with the UK National External Quality Assessment Scheme (UK NEQAS) Parasitology and with technical experts to launch a global NAAT EQA scheme in January 2017. METHODS: Panels of NAAT EQA specimens containing five major species of human-infecting Plasmodium at various parasite concentrations and negative samples were created in lyophilized blood (LB) and dried blood spot (DBS) formats. Two distributions per year were sent, containing five LB and five DBS specimens. Samples were tested and validated by six expert referee laboratories prior to distribution. Between 37 and 45 laboratories participated in each distribution and submitted results using the online submission portal of UK NEQAS. Participants were scored based on their laboratory's stated capacity to identify Plasmodium species, and individual laboratory reports were sent which included performance comparison with anonymized peers. RESULTS: Analysis of the first three distributions revealed that the factors that most significantly affected performance were sample format (DBS vs LB), species and parasite density, while laboratory location and the reported methodology used (type of nucleic acid extraction, amplification, or DNA vs RNA target) did not significantly affect performance. Referee laboratories performed better than non-referee laboratories. CONCLUSIONS: Globally, malaria NAAT assays now inform a range of clinical, epidemiological and research investigations. EQA schemes offer a way for laboratories to assess and improve their performance, which is critical to safeguarding the reliability of data and diagnoses especially in situations where various NAAT methodologies and protocols are in use.

Field evaluation of malaria malachite green loop-mediated isothermal amplification in health posts in Roraima state, Brazil.

BACKGROUND: Microscopic detection of malaria parasites is the standard method for clinical diagnosis of malaria in Brazil. However, malaria epidemiological surveillance studies specifically aimed at the detection of low-density infection and asymptomatic cases will require more sensitive and field-usable tools. The diagnostic accuracy of the colorimetric malachite green, loop-mediated, isothermal amplification (MG-LAMP) assay was evaluated in remote health posts in Roraima state, Brazil. METHODS: Study participants were prospectively enrolled from health posts (healthcare-seeking patients) and from nearby villages (healthy participants) in three different study sites. The MG-LAMP assay and microscopy were performed in the health posts. Two independent readers scored the MG-LAMP tests as positive (blue/green) or negative (clear). Sensitivity and specificity of local microscopy and MG-LAMP were calculated using results of PET-PCR as a reference. RESULTS: A total of 91 participants were enrolled. There was 100% agreement between the two MG-LAMP readers (Kappa = 1). The overall sensitivity and specificity of MG-LAMP were 90.0% (95% confidence interval (CI) 76.34-97.21%) and 94% (95% CI 83.76-98.77%), respectively. The sensitivity and specificity of local microscopy were 83% (95% CI 67.22-92.66%) and 100% (95% CI 93.02-100.00%), respectively. PET-PCR detected six mixed infections (infection with both Plasmodium falciparum and Plasmodium vivax); two of these were also detected by MG-LAMP and one by microscopy. Microscopy did not detect any Plasmodium infection in the 26 healthy participants; MG-LAMP detected Plasmodium in five of these and PET-PCR assay detected infection in three. Overall, performing the MG-LAMP in this setting did not present any particular challenges. CONCLUSION: MG-LAMP is a sensitive and specific assay that may be useful for the detection of malaria parasites in remote healthcare settings. These findings suggest that it is possible to implement simple molecular tests in facilities with limited resources.

Efficacy and safety of artemether-lumefantrine for the treatment of uncomplicated malaria and prevalence of Pfk13 and Pfmdr1 polymorphisms after a decade of using artemisinin-based combination therapy in mainland Tanzania.

BACKGROUND: The World Health Organization recommends regular therapeutic efficacy studies (TES) to monitor the performance of first and second-line anti-malarials. In 2016, efficacy and safety of artemether-lumefantrine (AL) for the treatment of uncomplicated falciparum malaria were assessed through a TES conducted between April and October 2016 at four sentinel sites of Kibaha, Mkuzi, Mlimba, and Ujiji in Tanzania. The study also assessed molecular markers of artemisinin and lumefantrine (partner drug) resistance. METHODS: Eligible patients were enrolled at the four sites, treated with standard doses of AL, and monitored for 28 days with clinical and laboratory assessments. The main outcomes were PCR corrected cure rates, day 3 positivity rates, safety of AL, and prevalence of single nucleotide polymorphisms in Plasmodium falciparum kelch 13 (Pfk13) (codon positions: 440-600) and P. falciparum multi-drug resistance 1 (Pfmdr1) genes (codons: N86Y, Y184F and D1246Y), markers of artemisinin and lumefantrine resistance, respectively. RESULTS: Of 344 patients enrolled, three withdrew, six were lost to follow-up; and results were analysed for 335 (97.4%) patients. Two patients had treatment failure (one early treatment failure and one recrudescent infection) after PCR correction, yielding an adequate clinical and parasitological response of > 98%. Day 3 positivity rates ranged from 0 to 5.7%. Common adverse events included cough, abdominal pain, vomiting, and diarrhoea. Two patients had serious adverse events; one died after the first dose of AL and another required hospitalization after the second dose of AL (on day 0) but recovered completely. Of 344 samples collected at enrolment (day 0), 92.7% and 100% were successfully sequenced for Pfk13 and Pfmdr1 genes, respectively. Six (1.9%) had non-synonymous mutations in Pfk13, none of which had been previously associated with artemisinin resistance. For Pfmdr1, the NFD haplotype (codons N86, 184F and D1246) was detected in 134 (39.0%) samples; ranging from 33.0% in Mlimba to 45.5% at Mkuzi. The difference among the four sites was not significant (p = 0.578). All samples had a single copy of the Pfmdr1 gene. CONCLUSION: The study indicated high efficacy of AL and the safety profile was consistent with previous reports. There were no known artemisinin-resistance Pfk13 mutations, but there was a high prevalence of a Pfmdr1 haplotype associated with reduced sensitivity to lumefantrine (but no reduced efficacy was observed in the subjects). Continued TES and monitoring of markers of resistance to artemisinin and partner drugs is critical for early detection of resistant parasites and to inform evidence-based malaria treatment policies. Trial Registration ClinicalTrials.gov NCT03387631.

Correction: Comparison of artemether-lumefantrine and chloroquine with and without primaquine for the treatment of Plasmodium vivax infection in Ethiopia: A randomized controlled trial.

[This corrects the article DOI: 10.1371/journal.pmed.1002299.].

Photo-Induced Electron Transfer Real-Time PCR for Detection of Plasmodium falciparum plasmepsin 2 Gene Copy Number.

Piperaquine is an important partner drug used in artemisinin-based combination therapies (ACTs). An increase in the plasmepsin 2 and 3 gene copy numbers has been associated with decreased susceptibility of Plasmodium falciparum to piperaquine in Cambodia. Here, we developed a photo-induced electron transfer real-time PCR (PET-PCR) assay to quantify the copy number of the P. falciparumplasmepsin 2 gene (PfPM2) that can be used in countries where P. falciparum is endemic to enhance molecular surveillance.

Next-Generation Sequencing and Bioinformatics Protocol for Malaria Drug Resistance Marker Surveillance.

The recent advances in next-generation sequencing technologies provide a new and effective way of tracking malaria drug-resistant parasites. To take advantage of this technology, an end-to-end Illumina targeted amplicon deep sequencing (TADS) and bioinformatics pipeline for molecular surveillance of drug resistance in P. falciparum, called malaria resistance surveillance (MaRS), was developed. TADS relies on PCR enriching genomic regions, specifically target genes of interest, prior to deep sequencing. MaRS enables researchers to simultaneously collect data on allele frequencies of multiple full-length P. falciparum drug resistance genes (crt, mdr1, k13, dhfr, dhps, and the cytochrome b gene), as well as the mitochondrial genome. Information is captured at the individual patient level for both known and potential new single nucleotide polymorphisms associated with drug resistance. The MaRS pipeline was validated using 245 imported malaria cases that were reported to the Centers for Disease Control and Prevention (CDC). The chloroquine resistance crt CVIET genotype (mutations underlined) was observed in 42% of samples, the highly pyrimethamine-resistant dhpsIRN triple mutant in 92% of samples, and the sulfadoxine resistance dhps mutation SGEAA in 26% of samples. The mdr1 NFSND genotype was found in 40% of samples. With the exception of two cases imported from Cambodia, no artemisinin resistance k13 alleles were identified, and 99% of patients carried parasites susceptible to atovaquone-proguanil. Our goal is to implement MaRS at the CDC for routine surveillance of imported malaria cases in the United States and to aid in the adoption of this system at participating state public health laboratories, as well as by global partners.

Emergence and Spread of kelch13 Mutations Associated with Artemisinin Resistance in Plasmodium falciparum Parasites in 12 Thai Provinces from 2007 to 2016.

Artemisinin-based combination therapy (ACT) is the most effective and widely used treatment for uncomplicated Plasmodium falciparum malaria and is a cornerstone for malaria control and prevention globally. Resistance to artemisinin derivatives has been confirmed in the Greater Mekong Subregion (GMS) and manifests as slow parasite clearance in patients and reduced ring stage susceptibility to artemisinins in survival assays. The P. falciparumkelch13 gene mutations associated with artemisinin-resistant parasites are now widespread in the GMS. We genotyped 277 samples collected during an observational study from 2012 to 2016 from eight provinces in Thailand to identify P. falciparum kelch13 mutations. The results were combined with previously reported genotyping results from Thailand to construct a map illustrating the evolution of P. falciparum kelch13 mutations from 2007 to 2016 in that country. Different mutant alleles were found in strains with different geographical origins. The artemisinin resistance-conferring Y493H and R539T mutations were detected mainly in eastern Thailand (bordering Cambodia), while P574L was found only in western Thailand and R561H only in northwestern Thailand. The C580Y mutation was found across the entire country and was nearing fixation along the Thai-Cambodia border. Overall, the prevalence of artemisinin resistance mutations increased over the last 10 years across Thailand, especially along the Thai-Cambodia border. Molecular surveillance and therapeutic efficacy monitoring should be intensified in the region to further assess the extent and spread of artemisinin resistance.

Investigation of a case of suspected transfusion-transmitted malaria.

BACKGROUND: Transfusion-transmitted malaria (TTM) is a rare occurrence with serious consequences for the recipient. A case study is presented as an example of best practices for conducting a TTM investigation. CASE REPORT: A 15-year-old male with a history of sickle cell disease developed fever after a blood transfusion. He was diagnosed with Plasmodium falciparum malaria and was successfully treated. The American Red Cross, New York State Department of Health, and the Centers for Disease Control and Prevention investigated the eight donors who provided components to the transfusion. The investigation to identify a malaria-positive donor included trace back of donors, serologic methods to identify donor(s) with a history of malaria exposure, polymerase chain reaction (PCR) testing, microsatellite analysis to identify the parasite in a donor and match its genotype to the parasite in the recipient, and reinterview of all donors to clarify malaria risk factors. RESULTS: One donor had evidence of infection with P. falciparum by PCR, elevated antibody titers, and previously undisclosed malaria risk factors. Reinterview revealed that the donor immigrated to the United States from Togo just short of 3 years before the blood donation. The donor was treated for asymptomatic low parasitemia infection. CONCLUSION: This investigation used standard procedures for investigating TTM but also demonstrated the importance of applying sensitive laboratory techniques to identify the infected donor, especially a donor with asymptomatic infection with low parasitemia. Repeat interview of all donors identified as having contributed to the transfused component provides complementary epidemiologic information to confirm the infected donor.

Prevalence of molecular markers of artemisinin and lumefantrine resistance among patients with uncomplicated Plasmodium falciparum malaria in three provinces in Angola, 2015.

BACKGROUND: Artemisinin-based combination therapy is the first-line anti-malarial treatment for uncomplicated Plasmodium falciparum infection in Angola. To date, the prevalence of polymorphisms in the pfk13 gene, associated with artemisinin resistance, and pfmdr1, associated with lumefantrine resistance, have not been systematically studied in Angola. METHODS: DNA was isolated from pretreatment and late treatment failure dried blood spots collected during the 2015 round of therapeutic efficacy studies in Benguela, Lunda Sul, and Zaire Provinces in Angola. The pfk13 propeller domain and pfmdr1 gene were sequenced and analysed for polymorphisms. Pfmdr1 copy number variation was assessed using a real-time PCR method. The association between pfmdr1 and pfk13 mutations and treatment failure was investigated. RESULTS: The majority of pretreatment (99%, 466/469) and all late treatment failure (100%, 50/50) samples were wild type for pfk13. Three of the pretreatment samples (1%) carried the A578S mutation commonly observed in Africa and not associated with artemisinin resistance. All 543 pretreatment and day of late treatment failure samples successfully analysed for pfmdr1 copy number variation carried one copy of pfmdr1. The NYD haplotype was the predominant pfmdr1 haplotype, present in 63% (308/491) of pretreatment samples, followed by NFD, which was present in 32% (157/491) of pretreatment samples. The pfmdr1 N86 allele was overrepresented in day of late treatment failure samples from participants receiving artemether-lumefantrine (p value 0.03). CONCLUSIONS: The pretreatment parasites in patients participating in therapeutic efficacy studies in 2015 in Angola's three sentinel sites showed genetic evidence of susceptibility to artemisinins, consistent with clinical outcome data showing greater than 99% day 3 clearance rates. The lack of increased pfmdr1 copy number is consistent with previous reports from sub-Saharan Africa. Although pfmdr1 NYD and NFD haplotypes were overrepresented in artemether-lumefantrine late treatment failure samples, their role as markers of resistance was unclear given that these haplotypes were also present in the majority of successfully treated patients in the artemether-lumefantrine treatment arms.

Dihydroartemisinin-piperaquine treatment failure in uncomplicated Plasmodium falciparum malaria case imported from Ethiopia.

Dihydroartemisinin-piperaquine (DHA-PPQ) is the artemisinin combination therapy that was recently introduced for the treatment of Plasmodium falciparum uncomplicated malaria, but emerging resistance in South-East Asia is threatening its use. This report describes a case of DHA-PPQ treatment failure in uncomplicated malaria occurring in an immigrant living in Italy, after a travel to Ethiopia. Thirty days after malaria recovery following DHA-PPQ therapy, the patient had malaria recrudescence. According to the genotyping analysis, the same P. falciparum was responsible for both episodes. Thus, it seems important to consider possible malaria recrudescence occurring after DHA-PPQ therapy in patients from African countries.