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.

Malaria remains a public health priority in Rwanda. The use of insecticide-treated nets (ITNs) is a key malaria prevention tool. However, expanding pyrethroid resistance threatens the gains made in malaria control. In 2018, the Rwandan malaria program strategic approach included the use of newer types of ITNs such as pyrethroid plus piperonyl butoxide (PBO) synergist-treated nets to counter pyrethroid resistance. In February 2020, 5,892,280 ITNs were distributed countrywide; 1,085,517 of these were PBO nets distributed in five districts. This study was a pragmatic observational study that leveraged the 2020 net distribution and routinely collected confirmed malaria cases to determine the impact of PBO nets 1 and 2 years after ITN distribution. No differences were observed in the average net coverage between the PBO and standard net districts. A significant reduction in malaria incidence was reported in both the PBO (P = 0.019) and two control districts that received standard nets (P = 0.008) 1 year after ITN distribution. However, 2 years after, this reduction was sustained only in the PBO (P = 0.013) and not in the standard net districts (P = 0.685). One year after net distribution, all districts had a significant reduction in malaria incidence rate (incidence rate ratio < 1). In the second year, incidence in districts with PBO nets continued to decrease, whereas in districts with standard nets, incidences were similar to predistribution levels. The results indicate that PBO nets are a promising tool to combat pyrethroid resistance in Rwanda, with protective effects of up to 2 years post distribution.

The vast majority of malaria mortality is attributed to one parasite species: Plasmodium falciparum. Asexual replication of the parasite within the red blood cell is responsible for the pathology of the disease. In Plasmodium, the endoplasmic reticulum (ER) is a central hub for protein folding and trafficking as well as stress response pathways. In this study, we tested the role of an uncharacterized ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localizes to the ER and is essential for asexual growth, specifically required for proper development of schizonts. PfGRP170 is essential for surviving heat shock, suggesting a critical role in cellular stress response. The data demonstrate that PfGRP170 interacts with the Plasmodium orthologue of the ER chaperone, BiP. Finally, we found that loss of PfGRP170 function leads to the activation of the Plasmodium eIF2α kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway.

Malaria is a debilitating parasitic disease that causes significant morbidity and mortality. Microscopic detection of parasites is currently the “gold standard” diagnostic. This technique is limited in its ability to detect low-density infections, is time consuming, and requires a highly trained microscopist. Malaria epidemiological surveillance studies especially aimed at the detection of low-density infection and asymptomatic cases will require more sensitive and user-friendly tools. We have shown previously that the molecular-based, colorimetric malachite green loop-mediated isothermal amplification (MG-LAMP) assay is a valuable tool for diagnosing malaria infection in a laboratory setting. In this study, we field evaluated this assay in a malaria diagnostic post in Roraima, Brazil. We prospectively collected 91 patient samples and performed microscopy, MG-LAMP, and real-time PCR (PET-PCR) to detect Plasmodium infection. Two independent readers were used to score the MG-LAMP tests to assess whether the sample was positive (blue/green) or negative (clear). There was 100% agreement between the two readers (Kappa=1). All tests detected 33 positive samples, but both the MG-LAMP and PET-PCR detected 6 and 7 more positive samples, respectively. The PET-PCR assay detected 6 mixed infections (defined as infection with both P. falciparum and P. vivax) while microscopy detected one and MG-LAMP detected two of these mixed infections. Microscopy did not detect any Plasmodium infection in 26 of the enrolled asymptomatic cases while MG-LAMP detected five and PET-PCR assay three positive cases. Overall, MG-LAMP provided a simpler and user-friendly molecular method for malaria diagnosis that is more sensitive than microscopy. Additionally, MG- LAMP has the capacity to test 38 samples per run (one hour), allowing for the screening of large number of samples which is appealing when large-scale studies are necessary e.g. in community surveillance studies. The current MG-LAMP assay was limited in its ability to detect mixed infection when compared to the PET-PCR, but otherwise proved to be a powerful tool for malaria parasite detection in the field and opens new perspectives in the implementation of surveillance studies in malaria elimination campaigns.

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.

PROBLEM/CONDITION: Malaria in humans is caused by intraerythrocytic protozoa of the genus Plasmodium. These parasites are transmitted by the bite of an infective female Anopheles species mosquito. Most malaria infections in the United States and its territories occur among persons who have traveled to regions with ongoing malaria transmission. However, among persons who have not traveled out of the country, malaria is occasionally acquired through exposure to infected blood or tissues, congenital transmission, nosocomial exposure, or local mosquitoborne transmission. Malaria surveillance in the United States and its territories provides information on its occurrence (e.g., temporal, geographic, and demographic), guides prevention and treatment recommendations for travelers and patients, and facilitates rapid transmission control measures if locally acquired cases are identified. PERIOD COVERED: This report summarizes confirmed malaria cases in persons with onset of illness in 2018 and trends in previous years. DESCRIPTION OF SYSTEM: Malaria cases diagnosed by blood smear microscopy, polymerase chain reaction, or rapid diagnostic tests are reported to local and state health departments through electronic laboratory reports or by health care providers or laboratory staff members directly reporting to CDC or health departments. Case investigations are conducted by local and state health departments, and reports are transmitted to CDC through the National Malaria Surveillance System (NMSS), the National Notifiable Diseases Surveillance System (NNDSS), or direct CDC clinical consultations. CDC reference laboratories provide diagnostic assistance and conduct antimalarial drug resistance marker testing on blood specimens submitted by health care providers or local or state health departments. This report summarizes data from the integration of all cases from NMSS and NNDSS, CDC clinical consultations, and CDC reference laboratory reports. RESULTS: CDC received reports of 1,823 confirmed malaria cases with onset of symptoms in 2018, including one cryptic case and one case acquired through a bone marrow transplant. The number of cases reported in 2018 is 15.6% fewer than in 2017. The number of cases diagnosed in the United States and its territories has been increasing since the mid-1970s; the number of cases reported in 2017 was the highest since 1972. Of the cases in 2018, a total of 1,519 (85.0%) were imported cases that originated from Africa; 1,061 (69.9%) of the cases from Africa were from West Africa, a similar proportion to what was observed in 2017. Among all cases, P. falciparum accounted for most infections (1,273 [69.8%]), followed by P. vivax (173 [9.5%]), P. ovale (95 [5.2%]), and P. malariae (48 [2.6%]). For the first time since 2008, an imported case of P. knowlesi was identified in the United States and its territories. Infections by two or more species accounted for 17 cases (<1.0%). The infecting species was not reported or was undetermined in 216 cases (11.9%). Most patients (92.6%) had symptom onset <90 days after returning to the United States or its territories from a country with malaria transmission. Of the U.S. civilian patients who reported reason for travel, 77.0% were visiting friends and relatives. Chemoprophylaxis with antimalarial medications are recommended for U.S. residents to prevent malaria while traveling in countries where it is endemic. Fewer U.S. residents with imported malaria reported taking any malaria chemoprophylaxis in 2018 (24.5%) than in 2017 (28.4%), and adherence was poor among those who took chemoprophylaxis. Among the 864 U.S. residents with malaria for whom information on chemoprophylaxis use and travel region were known, 95.0% did not adhere to or did not take a CDC-recommended chemoprophylaxis regimen. Among 683 women with malaria, 19 reported being pregnant. Of these, 11 pregnant women were U.S. residents, and one of whom reported taking chemoprophylaxis to prevent malaria but her adherence to chemoprophylaxis was not reported. Thirty-eight (2.1%) malaria cases occurred among U.S. military personnel in 2018, more than in 2017 (26 [1.2%]). Among all reported malaria cases in 2018, a total of 251 (13.8%) were classified as severe malaria illness, and seven persons died from malaria. In 2018, CDC analyzed 106 P. falciparum-positive and four P. falciparum mixed species specimens for antimalarial resistance markers (although certain loci were untestable in some specimens); identification of genetic polymorphisms associated with resistance to pyrimethamine were found in 99 (98.0%), to sulfadoxine in 49 (49.6%), to chloroquine in 50 (45.5%), and to mefloquine in two (2.0%); no specimens tested contained a marker for atovaquone or artemisinin resistance. INTERPRETATION: The importation of malaria reflects the overall trends in global travel to and from areas where malaria is endemic, and 15.6% fewer cases were imported in 2018 compared with 2017. Of imported cases, 59.3% were among persons who had traveled from West Africa. Among U.S. civilians, visiting friends and relatives was the most common reason for travel (77.1%). PUBLIC HEALTH ACTIONS: The best way for U.S. residents to prevent malaria is to take chemoprophylaxis medication before, during, and after travel to a country where malaria is endemic. Adherence to recommended malaria prevention strategies among U.S. travelers would reduce the number of imported cases. Reported reasons for nonadherence include prematurely stopping after leaving the area where malaria was endemic, forgetting to take the medication, and experiencing a side effect. Health care providers can make travelers aware of the risks posed by malaria and incorporate education to motivate them to be adherent to chemoprophylaxis. Malaria infections can be fatal if not diagnosed and treated promptly with antimalarial medications appropriate for the patient's age, pregnancy status, medical history, the likely country of malaria acquisition, and previous use of antimalarial chemoprophylaxis. Antimalarial use for chemoprophylaxis and treatment should be determined by the CDC guidelines, which are frequently updated. In April 2019, intravenous (IV) artesunate became the first-line medication for treatment of severe malaria in the United States and its territories. Artesunate was approved by the Food and Drug Administration (FDA) in 2020 and is commercially available (Artesunate for Injection) from major U.S. drug distributors (https://amivas.com). Stocking IV artesunate locally allows for immediate treatment of severe malaria once diagnosed and provides patients with the best chance of a complete recovery and no sequelae. With commercial IV artesunate now available, CDC will discontinue distribution of non-FDA-approved IV artesunate under an investigational new drug protocol on September 30, 2022. Detailed recommendations for preventing malaria are online at https://www.cdc.gov/malaria/travelers/drugs.html. Malaria diagnosis and treatment recommendations are also available online at https://www.cdc.gov/malaria/diagnosis_treatment. Health care providers who have sought urgent infectious disease consultation and require additional assistance on diagnosis and treatment of malaria can call the Malaria Hotline 9:00 a.m.-5:00 p.m. Eastern Time, Monday-Friday, at 770-488-7788 or 855-856-4713 or after hours for urgent inquiries at 770-488-7100. Persons submitting malaria case reports (care providers, laboratories, and state and local public health officials) should provide complete information because incomplete reporting compromises case investigations and public health efforts to prevent future infections and examine trends in malaria cases. Molecular surveillance of antimalarial drug resistance markers enables CDC to track, guide treatment, and manage drug resistance in malaria parasites both domestically and globally. A greater proportion of specimens from domestic malaria cases are needed to improve the completeness of antimalarial drug resistance analysis; therefore, CDC requests that blood specimens be submitted for any case of malaria diagnosed in the United States and its territories.

The Plasmodium falciparum protein VAR2CSA allows infected erythrocytes to accumulate within the placenta, inducing pathology and poor birth outcomes. Multiple exposures to placental malaria (PM) induce partial immunity against VAR2CSA, making it a promising vaccine candidate. However, the extent to which VAR2CSA genetic diversity contributes to immune evasion and virulence remains poorly understood. The deep sequencing of the var2csa DBL3X domain in placental blood from forty-nine primigravid and multigravid women living in malaria-endemic western Kenya revealed numerous unique sequences within individuals in association with chronic PM but not gravidity. Additional analysis unveiled four distinct sequence types that were variably present in mixed proportions amongst the study population. An analysis of the abundance of each of these sequence types revealed that one was inversely related to infant gestational age, another was inversely related to placental parasitemia, and a third was associated with chronic PM. The categorization of women according to the type to which their dominant sequence belonged resulted in the segregation of types as a function of gravidity: two types predominated in multigravidae whereas the other two predominated in primigravidae. The univariate logistic regression analysis of sequence type dominance further revealed that gravidity, maternal age, placental parasitemia, and hemozoin burden (within maternal leukocytes), reported a lack of antimalarial drug use, and infant gestational age and birth weight influenced the odds of membership in one or more of these sequence predominance groups. Cumulatively, these results show that unique var2csa sequences differentially appear in women with different PM exposure histories and segregate to types independently associated with maternal factors, infection parameters, and birth outcomes. The association of some var2csa sequence types with indicators of pathogenesis should motivate vaccine efforts to further identify and target VAR2CSA epitopes associated with maternal morbidity and poor birth outcomes. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

The state of Roraima, in Brazil, has recently seen an increase in the number of reported Plasmodium falciparum infections believed to be imported from neighboring countries. The objective of this study was to determine the prevalence of Plasmodium species among patients attending malaria health posts in Roraima and quantify the infections attributable to imported malaria. This cross-sectional case study was carried out between March 2016 and September 2018. Study participants were recruited as they exited the malaria health post. Information about residence, occupation and travel history was collected using a questionnaire. A dried blood spot was collected and used for malaria diagnosis by PCR. A total of 1222 patients were enrolled. Of the 80% Plasmodium positive samples, 50% were P. falciparum, 34% P. vivax, 8% mixed P. falciparum/P. vivax and 0.2% mixed P. falciparum/P. ovale infections and 8% tested positive for Plasmodium, but the species could not be identified. 80% of the malaria patients likely acquired infections in Venezuela and the remaining 20% acquired in Guyana, Brazil, Suriname and French Guyana. 50% of the study participants reported to be working in a mine. Results from this study support the hypothesis that imported malaria contribute to the bulk of malaria diagnosed in Roraima. These findings are in keeping with previous findings and should be considered when developing malaria control interventions.

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.

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.

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.

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.

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.

BACKGROUND: Recent studies show the first emergence of the R561H artemisinin-associated resistance marker in Africa, which highlights the importance of continued molecular surveillance to assess the selection and spread of this and other drug resistance markers in the region. METHOD: In this study, we used targeted deep amplicon sequencing (TADS) of 116 isolates collected in two areas of Cameroon to genotype the major drug resistance genes k13, crt, mdr1, dhfr, dhps, and the cytochrome b (cytb) in P. falciparum. RESULTS: No confirmed or associated artemisinin resistance markers were observed in Pfk13. In comparison, both major and minor alleles associated with drug resistance were found in Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps. Notably, a high frequency of other non-synonymous mutations was observed across all the genes, except Pfcytb, suggesting continued selection pressure. CONCLUSIONS: The results from this study support the continued use of artemisinin combination therapy (ACT) for treatment and administration of sulphadoxine-pyrimethamine for intermittent preventive therapy in pregnant women and for seasonal chemoprevention in these study sites in Cameroon.

BACKGROUND: Partial artemisinin resistance is suspected if delayed parasite clearance (ie, persistence of parasitaemia on day 3 after treatment initiation) is observed. Validated markers of artemisinin partial resistance in southeast Asia, Plasmodium falciparum kelch13 (Pfkelch13) R561H and P574L, have been reported in Rwanda but no association with parasite clearance has been observed. We aimed to establish the efficacy of artemether-lumefantrine and genetic characterisation of Pfkelch13 alleles and their association with treatment outcomes. METHODS: This open-label, single-arm, multicentre, therapeutic efficacy study was done in 2018 in three Rwandan sites: Masaka, Rukara, and Bugarama. Children aged 6-59 months with P falciparum monoinfection and fever were eligible and treated with a 3-day course of artemether-lumefantrine. Treatment response was monitored for 28 days using weekly microscopy screenings of blood samples for P falciparum. Mutations in Pfkelch13 and P falciparum multidrug resistance-1 (Pfmdr1) genes were characterised in parasites collected from enrolled participants. Analysis of flanking microsatellites surrounding Pfkelch13 was done to define the origins of the R561H mutations. The primary endpoint was PCR-corrected parasitological cure on day 28, as per WHO protocol. FINDINGS: 228 participants were enrolled and 224 (98·2%) reached the study endpoint. PCR-corrected efficacies were 97·0% (95% CI 88-100) in Masaka, 93·8% (85-98) in Rukara, and 97·2% (91-100) in Bugarama. Pfkelch13 R561H mutations were present in 28 (13%) of 218 pre-treatment samples and P574L mutations were present in two (1%) pre-treatment samples. 217 (90%) of the 240 Pfmdr1 haplotypes observed in the pretreatment samples, had either the NFD (N86Y, Y184F, D1246Y) or NYD haplotype. Eight (16%) of 51 participants in Masaka and 12 (15%) of 82 participants in Rukara were microscopically positive 3 days after treatment initiation, which was associated with pre-treatment presence of Pfkelch13 R561H in Masaka (p=0·0005). Genetic analysis of Pfkelch13 R561H mutations suggest their common ancestry and local origin in Rwanda. INTERPRETATION: We confirm evidence of emerging artemisinin partial resistance in Rwanda. Although artemether-lumefantrine remains efficacious, vigilance for decreasing efficacy, further characterisation of artemisinin partial resistance, and evaluation of additional antimalarials in Rwanda should be considered. FUNDING: The US President's Malaria Initiative. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

OBJECTIVE: In this study, the performance of a commercially available malaria LAMP assay (Alethia® Malaria Plus LAMP) was evaluated using retrospective clinical samples obtained from travelers returning to the United States of America (USA). Recently, several laboratories in non-malaria endemic countries evaluated the use of the loop mediated isothermal amplification (LAMP) assays for the diagnosis of imported malaria cases. These tests are simpler than polymerase-chain reaction (PCR)-based assays and were shown to have high sensitivity. Much of malaria diagnoses in the USA, is undertaken at the state level using mainly microscopy and rapid diagnostic tests (RDTs). However, molecular tools offer greater sensitivity over microscopy and RDTs. A reliable, easy to perform molecular assay can provide a test of choice for the accurate detection of malaria parasites in places where expert microscopy is lacking and/or for the detection of low-parasite density infections. RESULTS: The Alethia® Malaria Plus LAMP assay was easy to use, had similar test performances as the real-time PCR reference test and results were obtained faster (within 1 h) than the reference test. The sensitivity of the assay was 100% with a kappa score of 1 when compared to the reference PET-PCR assay.

In November 2018, we diagnosed a cluster of falciparum malaria cases in three Chilean travelers returning from Nigeria. Two patients were treated with sequential intravenous artesunate plus oral atovaquone/proguanil (AP) and one with oral AP. The third patient, a 23-year-old man, presented with fever on day 29 after oral AP treatment and was diagnosed with recrudescent falciparum malaria. The patient was then treated with oral mefloquine, followed by clinical recovery and resolution of parasitemia. Analysis of day 0 and follow-up blood samples, collected on days 9, 29, 34, 64, and 83, revealed that parasitemia had initially decreased but then increased on day 29. Sequencing confirmed Tyr268Cys mutation in the cytochrome b gene, associated with atovaquone resistance, in isolates collected on days 29 and 34 and P. falciparum dihydrofolate reductase mutation Asn51Ile, associated with proguanil resistance in all successfully sequenced samples. Molecular characterization of imported malaria contributes to clinical management in non-endemic countries, helps ascertain the appropriateness of antimalarial treatment policies, and contributes to the reporting of drug resistance patterns from endemic regions.

PROBLEM/CONDITION: Malaria in humans is caused by intraerythrocytic protozoa of the genus Plasmodium. These parasites are transmitted by the bite of an infective female Anopheles species mosquito. The majority of malaria infections in the United States occur among persons who have traveled to regions with ongoing malaria transmission. However, malaria is occasionally acquired by persons who have not traveled out of the country through exposure to infected blood products, congenital transmission, nosocomial exposure, or local mosquitoborne transmission. Malaria surveillance in the United States is conducted to provide information on its occurrence (e.g., temporal, geographic, and demographic), guide prevention and treatment recommendations for travelers and patients, and facilitate rapid transmission control measures if locally acquired cases are identified. PERIOD COVERED: This report summarizes confirmed malaria cases in persons with onset of illness in 2017 and trends in previous years. DESCRIPTION OF SYSTEM: Malaria cases diagnosed by blood film microscopy, polymerase chain reaction, or rapid diagnostic tests are reported to local and state health departments through electronic laboratory reports or by health care providers or laboratory staff members. Case investigations are conducted by local and state health departments, and reports are transmitted to CDC through the National Malaria Surveillance System (NMSS), the National Notifiable Diseases Surveillance System (NNDSS), or direct CDC consultations. CDC reference laboratories provide diagnostic assistance and conduct antimalarial drug resistance marker testing on blood samples submitted by health care providers or local or state health departments. This report summarizes data from the integration of all cases from NMSS and NNDSS, CDC reference laboratory reports, and CDC clinical consultations. RESULTS: CDC received reports of 2,161 confirmed malaria cases with onset of symptoms in 2017, including two congenital cases, three cryptic cases, and two cases acquired through blood transfusion. The number of malaria cases diagnosed in the United States has been increasing since the mid-1970s; in 2017, the number of cases reported was the highest in 45 years, surpassing the previous peak of 2,078 confirmed cases reported in 2016. Of the cases in 2017, a total of 1,819 (86.1%) were imported cases that originated from Africa; 1,216 (66.9%) of these came from West Africa. The overall proportion of imported cases originating from West Africa was greater in 2017 (57.6%) than in 2016 (51.6%). Among all cases, P. falciparum accounted for the majority of infections (1,523 [70.5%]), followed by P. vivax (216 [10.0%]), P. ovale (119 [5.5%]), and P. malariae (55 [2.6%]). Infections by two or more species accounted for 22 cases (1.0%). The infecting species was not reported or was undetermined in 226 cases (10.5%). CDC provided diagnostic assistance for 9.5% of confirmed cases and tested 8.0% of specimens with P. falciparum infections for antimalarial resistance markers. Most patients (94.8%) had symptom onset <90 days after returning to the United States from a country with malaria transmission. Of the U.S. civilian patients who reported reason for travel, 73.1% were visiting friends and relatives. The proportion of U.S. residents with malaria who reported taking any chemoprophylaxis in 2017 (28.4%) was similar to that in 2016 (26.4%), and adherence was poor among those who took chemoprophylaxis. Among the 996 U.S. residents with malaria for whom information on chemoprophylaxis use and travel region were known, 93.3% did not adhere to or did not take a CDC-recommended chemoprophylaxis regimen. Among 805 women with malaria, 27 reported being pregnant. Of these, 10 pregnant women were U.S. residents, and none reported taking chemoprophylaxis to prevent malaria. A total of 26 (1.2%) malaria cases occurred among U.S. military personnel in 2017, fewer than in 2016 (41 [2.0%]). Among all reported cases in 2017, a total of 312 (14.4%) were classified as severe malaria illnesses, and seven persons died. In 2017, CDC analyzed 117 P. falciparum-positive and six P. falciparum mixed-species samples for antimalarial resistance markers (although certain loci were untestable in some samples); identification of genetic polymorphisms associated with resistance to pyrimethamine were found in 108 (97.3%), to sulfadoxine in 77 (69.4%), to chloroquine in 38 (33.3%), to mefloquine in three (2.7%), and to atovaquone in three (2.7%); no specimens tested contained a marker for artemisinin resistance. The data completeness of key variables (species, country of acquisition, and resident status) was lower in 2017 (74.4%) than in 2016 (79.4%). INTERPRETATION: The number of reported malaria cases in 2017 continued a decades-long increasing trend, and for the second year in a row the highest number of cases since 1971 have been reported. Despite progress in malaria control in recent years, the disease remains endemic in many areas globally. The importation of malaria reflects the overall increase in global travel to and from these areas. Fifty-six percent of all cases were among persons who had traveled from West Africa, and among U.S. civilians, visiting friends and relatives was the most common reason for travel (73.1%). Frequent international travel combined with the inadequate use of prevention measures by travelers resulted in the highest number of imported malaria cases detected in the United States in 4 decades. PUBLIC HEALTH ACTIONS: The best way to prevent malaria is to take chemoprophylaxis medication during travel to a country where malaria is endemic. Adherence to recommended malaria prevention strategies among U.S. travelers would reduce the numbers of imported cases; reasons for nonadherence include prematurely stopping after leaving the area where malaria was endemic, forgetting to take the medication, and experiencing a side effect. Travelers might not understand the risk that malaria poses to them; thus, health care providers should incorporate risk education to motivate travelers to be adherent to chemoprophylaxis. Malaria infections can be fatal if not diagnosed and treated promptly with antimalarial medications appropriate for the patient's age, medical history, the likely country of malaria acquisition, and previous use of antimalarial chemoprophylaxis. Antimalarial use for chemoprophylaxis and treatment should be informed by the most recent guidelines, which are frequently updated. In 2018, two formulations of tafenoquine (i.e., Arakoda and Krintafel) were approved by the Food and Drug Administration (FDA) for use in the United States. Arakoda was approved for use by adults for chemoprophylaxis; the regimen requires a predeparture loading dose, taking the medication weekly during travel, and a short course posttravel. The Arakoda chemoprophylaxis regimen is shorter than alternative regimens, which could possibly improve adherence. This medication also might prevent relapses. Krintafel was approved for radical cure of P. vivax infections in those aged >16 years and should be co-administered with chloroquine (https://www.cdc.gov/malaria/new_info/2020/tafenoquine_2020.html). In April 2019, intravenous artesunate became the first-line medication for treatment of severe malaria in the United States. Artesunate was recently FDA approved but is not yet commercially available. The drug can be obtained from CDC under an investigational new drug protocol. Detailed recommendations for preventing malaria are available to the general public at the CDC website (https://www.cdc.gov/malaria/travelers/drugs.html). Health care providers should consult the CDC Guidelines for Treatment of Malaria in the United States and contact the CDC's Malaria Hotline for case management advice when needed. Malaria treatment recommendations are available online (https://www.cdc.gov/malaria/diagnosis_treatment) and from the Malaria Hotline (770-488-7788 or toll-free 855-856-4713). Persons submitting malaria case reports (care providers, laboratories, and state and local public health officials) should provide complete information because incomplete reporting compromises case investigations and efforts to prevent infections and examine trends in malaria cases. Molecular surveillance of antimalarial drug resistance markers (https://www.cdc.gov/malaria/features/ars.html) enables CDC to track, guide treatment, and manage drug resistance in malaria parasites both domestically and internationally. More samples are needed to improve the completeness of antimalarial drug resistance analysis; therefore, CDC requests that blood specimens be submitted for any case of malaria diagnosed in the United States.

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.

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.

The COVID-19 pandemic, caused by SARS-CoV-2, have surpassed 5 million cases globally. Current models suggest that low- and middle-income countries (LMICs) will have a similar incidence but substantially lower mortality rate than high-income countries. However, malaria and neglected tropical diseases (NTDs) are prevalent in LMICs, and coinfections are likely. Both malaria and parasitic NTDs can alter immunologic responses to other infectious agents. Malaria can induce a cytokine storm and pro-coagulant state similar to that seen in severe COVID-19. Consequently, coinfections with malaria parasites and SARS-CoV-2 could result in substantially worse outcomes than mono-infections with either pathogen, and could shift the age pattern of severe COVID-19 to younger age-groups. Enhancing surveillance platforms could provide signals that indicate whether malaria, NTDs, and COVID-19 are syndemics (synergistic epidemics). Based on the prevalence of malaria and NTDs in specific localities, efforts to characterize COVID-19 in LMICs could be expanded by adding testing for malaria and NTDs. Such additional testing would allow the determination of the rates of coinfection and comparison of severity of outcomes by infection status, greatly improving the understanding of the epidemiology of COVID-19 in LMICs and potentially helping to mitigate its impact.

Antimalarial drug resistance has historically arisen through convergent de novo mutations in Plasmodium falciparum parasite populations in Southeast Asia and South America. For the past decade in Southeast Asia, artemisinins, the core component of first-line antimalarial therapies, have experienced delayed parasite clearance associated with several pfk13 mutations, primarily C580Y. We report that mutant pfk13 has emerged independently in Guyana, with genome analysis indicating an evolutionary origin distinct from Southeast Asia. Pfk13 C580Y parasites were observed in 1.6% (14/854) of samples collected in Guyana in 2016-2017. Introducing pfk13 C580Y or R539T mutations by gene editing into local parasites conferred high levels of in vitro artemisinin resistance. In vitro growth competition assays revealed a fitness cost associated with these pfk13 variants, potentially explaining why these resistance alleles have not increased in frequency more quickly in South America. These data place local malaria control efforts at risk in the Guiana Shield. | All recommended treatments against malaria include a drug called artemisinin or some of its derivatives. However, there are concerns that Plasmodium falciparum, the parasite that causes most cases of malaria, will eventually develop widespread resistance to the drug. A strain of P. falciparum partially resistant to artemisinin was seen in Cambodia in 2008, and it has since spread across Southeast Asia. The resistance appears to be frequently linked to a mutation known as pfk13 C580Y. Southeast Asia and Amazonia are considered to be hotspots for antimalarial drug resistance, and the pfk13 C580Y mutation was detected in the South American country of Guyana in 2010. To examine whether the mutation was still circulating in this part of the world, Mathieu et al. collected and analyzed 854 samples across Guyana between 2016 and 2017. Overall, 1.6% of the samples had the pfk13 C580Y mutation, but this number was as high as 8.8% in one region. Further analyses revealed that the mutation in Guyana had not spread from Southeast Asia, but that it had occurred in Amazonia independently. To better understand the impact of the pfk13 C580Y mutation, Mathieu et al. introduced this genetic change into non-resistant parasites from a country neighbouring Guyana. As expected, the mutation made P. falciparum highly resistant to artemisinin, but it also slowed the growth rate of the parasite. This disadvantage may explain why the mutation has not spread more rapidly through Guyana in recent years. Artemisinin and its derivatives are always associated with other antimalarial drugs to slow the development of resistance; there are concerns that reduced susceptibility to artemisinin leads to the parasites becoming resistant to the partner drugs. Further research is needed to evaluate how the pfk13 C580Y mutation affects the parasite's response to the typical combination of drugs that are given to patients. | eng

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.

Laboratory detection of malaria antigens has proved valuable for research and epidemiological purposes. We recently developed a bead-based multiplex antigen assay for pan-Plasmodium and Plasmodium falciparum targets. Here, we report integration of a Plasmodium vivax-specific target to this multiplex panel: P. vivax lactate dehydrogenase (PvLDH). Within the multiplex panel, assay signal for purified PvLDH antigen titrated into the single-digit picogram range. Against a panel of PCR-confirmed samples from acute P. vivax infections (n = 36), sensitivity was 91.7% in using PvLDH detection for identifying the presence of parasites. Specificity against a panel of persons with no Plasmodium infection (n = 44) was 100%, and specificity against a panel of PCR-confirmed P. falciparum, Plasmodium malariae, or Plasmodium ovale infections (n = 164) was 90.2%. Addition of this PvLDH capture and detection system into the multiplex antigen panel will now allow for sensitive screening for species identification of both P. falciparum and P. vivax in the laboratory.

Artemether-lumefantrine (AL) is the first-line treatment for uncomplicated Plasmodium falciparum infection in Colombia. To assess AL efficacy for uncomplicated falciparum malaria in Quibdo, Choco, Colombia, we conducted a 28-day therapeutic efficacy study (TES) following the WHO guidelines. From July 2018 to February 2019, febrile patients aged 5-65 years with microscopy-confirmed P. falciparum mono-infection and asexual parasite density of 250-100,000 parasites/microL were enrolled and treated with a supervised 3-day course of AL. The primary endpoint was adequate clinical and parasitological response (ACPR) on day 28. We attempted to use polymerase chain reaction (PCR) genotyping to differentiate reinfection and recrudescence, and conducted genetic testing for antimalarial resistance-associated genes. Eighty-eight patients consented and were enrolled: four were lost to follow-up or missed treatment doses. Therefore, 84 (95.5%) participants reached a valid endpoint: treatment failure or ACPR. No patient remained microscopy positive for malaria on day 3, evidence of delayed parasite clearance and artemisinin resistance. One patient had recurrent infection (12 parasites/microL) on day 28. Uncorrected ACPR rate was 98.8% (83/84) (95% CI: 93.5-100%). The recurrent infection sample did not amplify during molecular testing, giving a PCR-corrected ACPR of 100% (83/83) (95% CI: 95.7-100%). No P. falciparum kelch 13 polymorphisms associated with artemisinin resistance were identified. Our results support high AL efficacy for falciparum malaria in Choco. Because of the time required to conduct TESs in low-endemic settings, it is important to consider complementary alternatives to monitor antimalarial efficacy and resistance.

Given that the C580Y polymorphism in the Plasmodium falciparum propeller domain of the kelch 13 gene (pfk13) was documented in Guyana, monitoring for mutations associated with antimalarial resistance was undertaken in neighboring Roraima state in Brazil. Polymorphisms in the pfmdr1 and pfk13 genes were investigated in 275 P. falciparum samples. No pfk13 mutations were observed. Triple mutants 184F, 1042D, and 1246Y were observed in 100% of the samples successfully sequenced for the pfmdr1 gene, with 20.1% of these having an additional mutation at codon 1034C. Among them, 2.5% of samples harbored two copies of the pfmdr1 gene. We found no evidence of the spread of C580Y parasites to Roraima state, Brazil. As previously observed, the 184F, 1042D, and 1246Y mutations in the pfmdr1 gene appear to be fixed in this region. Continued molecular surveillance is essential to detect any potential migration or local emergence of artemisinin-resistant mutation.

PROBLEM/CONDITION: Malaria in humans is caused by intraerythrocytic protozoa of the genus Plasmodium. These parasites are transmitted by the bite of an infective female Anopheles species mosquito. The majority of malaria infections in the United States occur among persons who have traveled to regions with ongoing malaria transmission. However, malaria is occasionally acquired by persons who have not traveled out of the country through exposure to infected blood products, congenital transmission, laboratory exposure, or local mosquitoborne transmission. Malaria surveillance in the United States is conducted to provide information on its occurrence (e.g., temporal, geographic, and demographic), guide prevention and treatment recommendations for travelers and patients, and facilitate transmission control measures if locally acquired cases are identified. PERIOD COVERED: This report summarizes confirmed malaria cases in persons with onset of illness in 2016 and summarizes trends in previous years. DESCRIPTION OF SYSTEM: Malaria cases diagnosed by blood film microscopy, polymerase chain reaction, or rapid diagnostic tests are reported to local and state health departments by health care providers or laboratory staff members. Case investigations are conducted by local and state health departments, and reports are transmitted to CDC through the National Malaria Surveillance System (NMSS), the National Notifiable Diseases Surveillance System (NNDSS), or direct CDC consultations. CDC reference laboratories provide diagnostic assistance and conduct antimalarial drug resistance marker testing on blood samples submitted by health care providers or local or state health departments. This report summarizes data from the integration of all NMSS and NNDSS cases, CDC reference laboratory reports, and CDC clinical consultations. RESULTS: CDC received reports of 2,078 confirmed malaria cases with onset of symptoms in 2016, including two congenital cases, three cryptic cases, and one case acquired through blood transfusion. The number of malaria cases diagnosed in the United States has been increasing since the mid-1970s. However, in 2015 a decrease occurred in the number of cases, specifically from the region of West Africa, likely due to altered travel related to the Ebola virus disease outbreak. The number of confirmed malaria cases in 2016 represents a 36% increase compared with 2015, and the 2016 total is 153 more cases than in 2011, which previously had the highest number of cases (1,925 cases). In 2016, a total of 1,729 cases originated from Africa, and 1,061 (61.4%) of these came from West Africa. P. falciparum accounted for the majority of the infections (1,419 [68.2%]), followed by P. vivax (251 [12.1%]). Fewer than 2% of patients were infected by two species (23 [1.1%]). The infecting species was not reported or was undetermined in 10.8% of cases. CDC provided diagnostic assistance for 12.1% of confirmed cases and tested 10.8% of specimens with P. falciparum infections for antimalarial resistance markers. Of the U.S. resident patients who reported reason for travel, 69.4% were travelers who were visiting friends and relatives. The proportion of U.S. residents with malaria who reported taking any chemoprophylaxis in 2016 (26.3%) was similar to that in 2015 (26.6%), and adherence was poor among those who took chemoprophylaxis. Among the 964 U.S. residents with malaria for whom information on chemoprophylaxis use and travel region were known, 94.0% of patients with malaria did not adhere to or did not take a CDC-recommended chemoprophylaxis regimen. Among 795 women with malaria, 50 were pregnant, and one had adhered to mefloquine chemoprophylaxis. Forty-one (2.0%) malaria cases occurred among U.S. military personnel in 2016, a comparable proportion to that in 2015 (23 cases [1.5%]). Among all reported cases in 2016, a total of 306 (14.7%) were classified as severe illnesses, and seven persons died. In 2016, CDC analyzed 144 P. falciparum-positive and nine P. falciparum mixed species samples for surveillance of antimalarial resistance markers (although certain loci were untestable in some samples); genetic polymorphisms associated with resistance to pyrimethamine were identified in 142 (97.9%), to sulfadoxine in 98 (70.5%), to chloroquine in 67 (44.7%), to mefloquine in six (4.3%), and to atovaquone in one (<1.0%). The completeness of key variables (e.g., species, country of acquisition, and resident status) was 79.4% in 2016 and 75.7% in 2015. INTERPRETATION: The number of reported malaria cases in 2016 continued a decades-long increasing trend and is the highest since 1972. The importation of malaria reflects the overall increase in global travel trends to and from areas where malaria is endemic; a transient decrease in the acquisition of cases, predominantly from West Africa, occurred in 2015. In 2016, more cases (absolute number) originated from regions of the world with widespread malaria transmission. Since the early 2000s, worldwide interventions to reduce malaria have been successful; however, progress has plateaued in recent years, the disease remains endemic in many regions, and the use of appropriate prevention measures by travelers remains inadequate. PUBLIC HEALTH ACTIONS: The best way to prevent malaria is to take chemoprophylaxis medication during travel to a country where malaria is endemic. Malaria infections can be fatal if not diagnosed and treated promptly with antimalarial medications appropriate for the patient's age and medical history, the likely country of malaria acquisition, and previous use of antimalarial chemoprophylaxis. In 2018, two tafenoquine-based antimalarials were approved by the Food and Drug Administration (FDA) for use in the United States. Arakoda was approved for use by adults for chemoprophylaxis and is available as a weekly dosage that is convenient during travel, which might improve adherence and also can prevent relapses from P. vivax and P. ovale infections. Krintafel was approved for radical cure of P. vivax infections in those >16 years old. In April 2019, intravenous artesunate became the first-line medication for treatment of severe malaria in the United States. Because intravenous artesunate is not FDA approved, it is available from CDC under an investigational new drug protocol. Detailed recommendations for preventing malaria are available to the general public at the CDC website (https://www.cdc.gov/malaria/travelers/drugs.html). Health care providers should consult the CDC Guidelines for Treatment of Malaria in the United States and contact the CDC's Malaria Hotline for case management advice when needed. Malaria treatment recommendations are available online (https://www.cdc.gov/malaria/diagnosis_treatment) and from the Malaria Hotline (770-488-7788 or toll-free at 855-856-4713). Persons submitting malaria case reports (care providers, laboratories, and state and local public health officials) should provide complete information because incomplete reporting compromises case investigations and efforts to prevent infections and examine trends in malaria cases. Adherence to recommended malaria prevention strategies is low among U.S. travelers; reasons for nonadherence include prematurely stopping after leaving the area where malaria was endemic, forgetting to take the medication, and experiencing a side effect. Molecular surveillance of antimalarial drug resistance markers (https://www.cdc.gov/malaria/features/ars.html) enables CDC to track, guide treatment, and manage drug resistance in malaria parasites both domestically and internationally. More samples are needed to improve the completeness of antimalarial drug resistance analysis; therefore, CDC requests that blood specimens be submitted for all cases of malaria diagnosed in the United States.

The vast majority of malaria mortality is attributed to one parasite species: Plasmodium falciparum. Asexual replication of the parasite within the red blood cell is responsible for the pathology of the disease. In Plasmodium, the endoplasmic reticulum (ER) is a central hub for protein folding and trafficking as well as stress response pathways. In this study, we tested the role of an uncharacterized ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localizes to the ER and is essential for asexual growth, specifically required for proper development of schizonts. PfGRP170 is essential for surviving heat shock, suggesting a critical role in cellular stress response. The data demonstrate that PfGRP170 interacts with the Plasmodium orthologue of the ER chaperone, BiP. Finally, we found that loss of PfGRP170 function leads to the activation of the Plasmodium eIF2alpha kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway.

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.

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.