We thank Rasmussen and Ringwald for further highlighting the importance of routine monitoring of antimalarial drug efficacy in sub-Saharan Africa, including Angola (1). Longitudinal monitoring is critical to identify potential new, persistent, and/or expanding foci of parasite resistance to available drugs. In 3 of the last 4 rounds, artemether-lumefantrine (AL) was estimated to have an efficacy of <90% at one of the three sentinel sites in Angola. To our knowledge, in sub-Saharan Africa, only Angola and Burkina Faso (2) have shown AL efficacy of <90% across multiple therapeutic efficacy study (TES) rounds. Thus, we chose a title to highlight this persistent concern. | | We concur that the significance of the high rates of day 2 slide positivity in Lunda Sul Province is not fully known, and as pointed out, there may be various explanations for this finding. Measuring drug levels is resource intensive and not feasible every year, but this could help rule out underdosing in future studies. However, we believe our study procedures, as described in this and previous studies, are robust and thus make systematic underdosing unlikely. We have always strictly adhered to WHO guidelines, including hemoglobin criteria and analysis of day 1 severe cases, to inform our classifications.

Monitoring antimalarial efficacy is important to detect the emergence of parasite drug resistance. Angola conducts in vivo therapeutic efficacy studies (TESs) every 2 years in its fixed sentinel sites in Benguela, Lunda Sul, and Zaire provinces. Children with uncomplicated Plasmodium falciparum malaria were treated with artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ), dihydroartemisinin-piperaquine (DP), or artesunate-pyronaridine (ASPY) and followed for 28 (AL and ASAQ) or 42 days (DP and ASPY) to assess clinical and parasitological response to treatment. Two drugs were sequentially assessed in each site in February-July 2021. The primary indicator was the Kaplan-Meier estimate of the PCR-corrected efficacy at the end of the follow-up period. A total of 622 patients were enrolled in the study and 590 (95%) participants reached a study endpoint. By day 3, ≥98% of participants were slide-negative in all study sites and arms. After PCR correction, day 28 AL efficacy was 88.0% (95% CI: 82%-95%) in Zaire and 94.7% (95% CI: 90%-99%) in Lunda Sul. For ASAQ, day 28 efficacy was 92.0% (95% CI: 87%-98%) in Zaire and 100% in Lunda Sul. Corrected day 42 efficacy was 99.6% (95% CI: 99%-100%) for ASPY and 98.3% (95% CI: 96%-100%) for DP in Benguela. High day 3 clearance rates suggest no clinical evidence of artemisinin resistance. This was the fourth of five rounds of TES in Angola showing a corrected AL efficacy <90% in a site. For Zaire, AL has had an efficacy <90% in 2013, 2015, and 2021. ASAQ, DP, and ASPY are appropriate choices as artemisinin-based combination therapies in Angola.

Sulfadoxine-pyrimethamine (SP) is used for prevention of malaria in pregnant women in Angola. We sequenced the Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes, implicated in SP resistance, in samples collected during a 2019 study of artemisinin-based combination therapy efficacy in Benguela, Lunda Sul, and Zaire provinces. A total of 90 day 0 and day of failure samples were individually sequenced, while 508 day 0 samples from participants without recurrent parasitemia were pooled after DNA extraction into 61 pools. The N51I, C59R, and S108N pfdhfr mutations and A437G pfdhps mutations were present at high proportions in all provinces (weighted allele frequencies, 62% to 100%). The K540E pfdhps mutation was present at lower proportions (10% to 14%). The A581G pfdhps mutation was only observed in Zaire, at a 4.6% estimated prevalence. The I431V and A613S mutations were also only observed in Zaire, at a prevalence of 2.8% to 2.9%. The most common (27% to 66%) reconstructed haplotype in all three provinces was the canonical quadruple pfdhfr pfdhps mutant. The canonical quintuple mutant was absent in Lunda Sul and Benguela and present in 7.9% of samples in Zaire. A single canonical sextuple (2.6%) mutant was observed in Zaire Province. Proportions of the pfdhps K540E and A581G mutations were well below the World Health Organization thresholds for meaningful SP resistance (prevalence of 95% for K540E and 10% for A581G). Samples from therapeutic efficacy studies represent a convenient source of samples for monitoring SP resistance markers.

Immunoglobulin (Ig) production during and after infection with Plasmodium parasites is one of the greatest adaptive immune defenses the human host has against this parasite. Infection with P. falciparum has been shown to induce different B cell maturation responses dependent upon the age of the patient, number of previous exposures, and severity of the disease. Described here are dynamics of Ig responses to a panel of 32 P. falciparum antigens by patients followed for 42 days and classified individuals as showing characteristics of an apparent first P. falciparum infection (nave) or a repeat exposure (non-nave). Six parameters were modeled to characterize the dynamics of IgM, IgG(1), IgG(3), and IgA for these two exposure groups with differences assessed among Ig isotypes/subclasses and unique antigens. Nave patients had significantly longer periods of time to reach peak Ig titer (range 4-7 days longer) and lower maximum Ig titers when compared with non-nave patients. Modeled time to seronegativity was significantly higher in non-nave patients for IgM and IgA, but not for the two IgG subclasses. IgG(1) responses to Rh2030, HSP40, and PfAMA1 were at the highest levels for non-nave participants and may be used to predict previous or nascent exposure by themselves. The analyses presented here demonstrate the differences in the development of the Ig response to P. falciparum if the infection represents a boosting response or a primary exposure. Consistency in Ig isotype/subclasses estimates and specific data for P. falciparum antigens can better guide interpretation of seroepidemiological data among symptomatic persons.

The majority of symptomatic malaria in sub-Saharan Africa is caused by Plasmodium falciparum. Infection with Plasmodium ovale is often not recorded and not considered clinically relevant. Here, we describe 8 cases of P ovale infection from 3 African countries-all of which were misdiagnosed at the presenting health facility.

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.

Human Plasmodium infection produces a robust adaptive immune response. Time courses for 104 children followed for 42 days after initiation of Plasmodium falciparum chemotherapy were assayed for antibody levels to the five isotypes of human immunoglobulins (Ig) and 4 subclasses of IgG for 32 P. falciparum antigens encompassing all 4 parasite stages of human infection. IgD and IgE against these antigens were undetectable at 1:100 serum concentration, but other Ig isotypes and IgG subclasses were consistently observed against all antigens. Five quantitative parameters were developed to directly compare Ig response among isotypes and antigens: C(max), maximum antibody level; Δ(C), difference between C(max) and the antibody level at Day 0; t(max), time in days to reach C(max); t(1/2), Ig signal half-life in days; t(neg), estimated number of days until complete loss of Ig signal. Classical Ig patterns for a bloodborne pathogen were seen with IgM showing early t(max) and IgG production highest among Ig isotypes. However, some unexpected trends were observed such as IgA showing a biphasic pattern for many antigens. Variability among these dynamics of Ig acquisition and loss was noted for different P. falciparum antigens and able to be compared both quantitatively and statistically. This parametrization methodology allows direct comparison of Ig isotypes produced against various Plasmodium antigens following malaria infection, and the same methodology could be applied to other longitudinal serologic studies from P. falciparum or different pathogens. Specifically for P. falciparum seroepidemiological studies, reliable and quantitative estimates regarding the IgG dynamics in human populations can better optimize modeling efforts for serological outputs.

BACKGROUND: Biennial therapeutic efficacy monitoring is a crucial activity for ensuring efficacy of currently used artemisinin-based combination therapy in Angola. METHODS: Children with acute uncomplicated P. falciparum infection in sentinel sites in Benguela, Zaire, and Lunda Sul Provinces were treated with artemether-lumefantrine (AL) or artesunate amodiaquine (ASAQ) and followed for 28 days to assess clinical and parasitological response. Molecular correction was performed using seven microsatellite markers. Samples from treatment failures were genotyped for the pfk13, pfcrt, and pfmdr1 genes. RESULTS: Day 3 clearance rates were ≥95% in all arms. Uncorrected Day-28 Kaplan-Meier efficacy estimates ranged from 84.2 to 90.1% for the AL arms, and 84.7 to 100% for the ASAQ arms. Corrected Day-28 estimates were 87.6% (95% Confidence interval [CI]: 81-95%) for the AL arm in Lunda Sul, 92.2% (95%CI: 87-98%) for AL in Zaire, 95.6% (95%CI: 91-100%) for ASAQ in Zaire, 98.4% (95%CI: 96-100%) for AL in Benguela, and 100% for ASAQ in Benguela and Lunda Sul. All 103 analyzed samples had wildtype pfk13 sequences. The 76T pfcrt allele was found in most (92%, 11/12) ASAQ late failure samples but only 16% (4/25) of AL failure samples. The N86 pfmdr1 allele was found in 97% (34/35) of treatment failures. CONCLUSION: AL efficacy in Lunda Sul was below the 90% World Health Organization threshold, the third time in four rounds that this threshold was crossed for an AL arm in Angola. In contrast, observed ASAQ efficacy has not been below 95% to date in Angola, including this latest round.

BACKGROUND: Lingering post-treatment parasite antigen in blood complicates malaria diagnosis through antigen detection. Characterization of antigen clearance dynamics is important for interpretation of positive antigen detection tests. RESULTS: We used a bead-based serological assay to measure lactate dehydrogenase (LDH), aldolase (Aldo), and histidine-rich protein 2 (HRP2) levels in 196 children with Plasmodium falciparum malaria treated with effective antimalarials and followed for 28 to 42 days as part of therapeutic efficacy studies in Angola. Compared to pre-treatment levels, antigen concentrations two days after treatment declined by 99.7% for LDH, 96.3% for Aldo, and 54.6% for HRP2. After Day 2, assuming a first-order kinetics clearance model, half-lives of the antigens were 1.8 days (95% CI: 1.5-2.3) for LDH, 3.2 days (95% CI: 3.0-3.4) for Aldo, and 4.8 days (95% CI: 4.7-4.9) for HRP2. CONCLUSIONS: LDH and Aldo show substantially different clearance rates than HRP2, and their presence is largely indicative of active infection.

BACKGROUND: The Angolan government recommends three artemisinin-based combinations for the treatment of uncomplicated Plasmodium falciparum malaria: artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ), and dihydroartemisinin-piperaquine (DP). Due to the threat of emerging anti-malarial drug resistance, it is important to periodically monitor the efficacy of artemisinin-based combination therapy (ACT). This study evaluated these medications' therapeutic efficacy in Benguela, Lunda Sul, and Zaire Provinces. METHODS: Enrollment occurred between March and July 2017. Study participants were children with P. falciparum monoinfection from each provincial capital. Participants received a 3-day course of a quality-assured artemisinin-based combination and were monitored for 28 (AL and ASAQ arms) or 42 days (DP arm). Each ACT was assessed in two provinces. The primary study endpoints were: (1) follow-up without complications and (2) failure to respond to treatment or development of recurrent P. falciparum infection. Parasites from each patient experiencing recurrent infection were genotyped to differentiate new infection from recrudescence of persistent parasitaemia. These parasites were also analysed for molecular markers associated with ACT resistance. RESULTS: Of 608 children enrolled in the study, 540 (89%) reached a primary study endpoint. Parasitaemia was cleared within 3 days of medication administration in all participants, and no early treatment failures were observed. After exclusion of reinfections, the corrected efficacy of AL was 96% (91-100%, 95% confidence interval) in Zaire and 97% (93-100%) in Lunda Sul. The corrected efficacy of ASAQ was 100% (97-100%) in Benguela and 93% (88-99%) in Zaire. The corrected efficacy of DP was 100% (96-100%) in Benguela and 100% in Lunda Sul. No mutations associated with artemisinin resistance were identified in the pfk13 gene in the 38 cases of recurrent P. falciparum infection. All 33 treatment failures in the AL and ASAQ arms carried pfmdr1 or pfcrt mutations associated with lumefantrine and amodiaquine resistance, respectively, on day of failure. CONCLUSIONS: AL, ASAQ, and DP continue to be efficacious against P. falciparum malaria in these provinces of Angola. Rapid parasite clearance and the absence of genetic evidence of artemisinin resistance are consistent with full susceptibility to artemisinin derivatives. Periodic monitoring of in vivo drug efficacy remains a priority routine activity for Angola.

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.

Antimalarial drug resistance is an evolving global health security threat to malaria control. Early detection of Plasmodium falciparum resistance through therapeutic efficacy studies and associated genetic analyses may facilitate timely implementation of intervention strategies. The US President's Malaria Initiative-supported Antimalarial Resistance Monitoring in Africa Network has assisted numerous laboratories in partner countries in acquiring the knowledge and capability to independently monitor for molecular markers of antimalarial drug resistance.

Background: Response to antimalarial treatment is assessed using serial microscopy. New techniques for accurate measurement of the Plasmodium falciparum histidine rich protein 2 (HRP2) antigen have made monitoring antigen concentration over time a potential alternative for assessing treatment response. Methods: Post-treatment HRP2 concentration was measured in longitudinal samples from 537 participants with P. falciparum malaria from efficacy trials in Angola, Tanzania, and Senegal. The HRP2 half-life was estimated using a first-order kinetics clearance model. The association between HRP2 concentration three days post-treatment and recrudescence of infection was assessed. Results: Despite substantial variation in HRP2 concentration at baseline, HRP2 concentration in patients consistently showed a first-order exponential decline. The median half-life of HRP2 was estimated to be 4.5 days (interquartile range: 3.3-6.6) in Angola, 4.7 days (4.0-5.9) in Tanzania, and 3.0 days (2.1-4.5) in Senegal. The day 3 HRP2 concentration was predictive of eventual recrudescence, with an area under the receiver operating characteristic curve of 0.86 (95% confidence interval: 0.73-0.99). Conclusions: Consistent HRP2 clearance dynamics following successful antimalarial treatment imply a common underlying biological clearance mechanism. Patients that ultimately failed treatment did not exhibit this same pattern of clearance, even in the absence of other indications of inadequate response to treatment.

Most malaria testing is by rapid diagnostic tests (RDTs) that detect Plasmodium falciparum histidine-rich protein 2 (HRP2). Recently, several RDT manufacturers have developed highly sensitive RDTs (hsRDTs), promising a limit of detection (LOD) orders of magnitude lower than conventional RDTs. To model the added utility of hsRDTs, HRP2 concentration in Angolan outpatients was measured quantitatively using an ultrasensitive bead-based assay. The distribution of HRP2 concentration was bimodal in both afebrile and febrile patients. The conventional RDT was able to detect 81% of all HRP2-positive febrile patients and 52-77% of HRP2-positive afebrile patients. The added utility of hsRDTs was estimated to be greater in afebrile patients, where an hsRDT with a LOD of 200 pg/mL would detect an additional 50-60% of HRP2-positive persons compared with a conventional RDT with a LOD of 3,000 pg/mL. In febrile patients, the hsRDT would detect an additional 10-20% of cases. Conventional RDTs already capture the vast majority of symptomatic HRP2-positive individuals, and hsRDTs would have to reach a sufficiently low LOD approaching 200 pg/mL to provide added utility in identifying HRP2-positive, asymptomatic individuals.

BACKGROUND: Malaria accounts for the largest portion of healthcare demand in Angola. A pillar of malaria control in Angola is the appropriate management of malaria illness, including testing of suspect cases with rapid diagnostic tests (RDTs) and treatment of confirmed cases with artemisinin-based combination therapy (ACT). Periodic systematic evaluations of malaria case management are recommended to measure health facility readiness and adherence to national case management guidelines. METHODS: Cross-sectional health facility surveys were performed in low-transmission Huambo and high-transmission Uige Provinces in early 2016. In each province, 45 health facilities were randomly selected from among all public health facilities stratified by level of care. Survey teams performed inventories of malaria commodities and conducted exit interviews and re-examinations, including RDT testing, of a random selection of all patients completing outpatient consultations. Key health facility readiness and case management indicators were calculated adjusting for the cluster sampling design and utilization. RESULTS: Availability of RDTs or microscopy on the day of the survey was 71% (54-83) in Huambo and 85% (67-94) in Uige. At least one unit dose pack of one formulation of an ACT (usually artemether-lumefantrine) was available in 83% (66-92) of health facilities in Huambo and 79% (61-90) of health facilities in Uige. Testing rates of suspect malaria cases in Huambo were 30% (23-38) versus 69% (53-81) in Uige. Overall, 28% (13-49) of patients with uncomplicated malaria, as determined during the re-examination, were appropriately treated with an ACT with the correct dose in Huambo, compared to 60% (42-75) in Uige. Incorrect case management of suspect malaria cases was associated with lack of healthcare worker training in Huambo and ACT stock-outs in Uige. CONCLUSIONS: The results reveal important differences between provinces. Despite similar availability of testing and ACT, testing and treatment rates were lower in Huambo compared to Uige. A majority of true malaria cases seeking care in health facilities in Huambo were not appropriately treated with anti-malarials, highlighting the importance of continued training and supervision of healthcare workers in malaria case management, particularly in areas with decreased malaria transmission.

BACKGROUND: Recent anti-malarial resistance monitoring in Angola has shown efficacy of artemether-lumefantrine (AL) in certain sites approaching the key 90% lower limit of efficacy recommended for artemisinin-based combination therapy. In addition, a controversial case of malaria unresponsive to artemisinins was reported in a patient infected in Lunda Sul Province in 2013. METHODS: During January-June 2015, investigators monitored the clinical and parasitological response of children with uncomplicated Plasmodium falciparum infection treated with AL, artesunate-amodiaquine (ASAQ), or dihydroartemisinin-piperaquine (DP). The study comprised two treatment arms in each of three provinces: Benguela (AL, ASAQ), Zaire (AL, DP), and Lunda Sul (ASAQ, DP). Samples from treatment failures were analysed for molecular markers of resistance for artemisinin (K13) and lumefantrine (pfmdr1). RESULTS: A total of 467 children reached a study endpoint. Fifty-four treatment failures were observed: four early treatment failures, 40 re-infections and ten recrudescences. Excluding re-infections, the 28-day microsatellite-corrected efficacy was 96.3% (95% CI 91-100) for AL in Benguela, 99.9% (95-100) for ASAQ in Benguela, 88.1% (81-95) for AL in Zaire, and 100% for ASAQ in Lunda Sul. For DP, the 42-day corrected efficacy was 98.8% (96-100) in Zaire and 100% in Lunda Sul. All treatment failures were wild type for K13, but all AL treatment failures had pfmdr1 haplotypes associated with decreased lumefantrine susceptibility. CONCLUSIONS: No evidence was found to corroborate the specific allegation of artemisinin resistance in Lunda Sul. The efficacy below 90% of AL in Zaire matches findings from 2013 from the same site. Further monitoring, particularly including measurement of lumefantrine blood levels, is recommended.

Infections with the malaria parasite Plasmodium falciparum typically comprise multiple strains, especially in high-transmission areas where infectious mosquito bites occur frequently. However, little is known about the dynamics of mixed-strain infections, particularly whether strains sharing a host compete or grow independently. Competition between drug-sensitive and drug-resistant strains, if it occurs, could be a crucial determinant of the spread of resistance. We analysed 1341 P. falciparum infections in children from Angola, Ghana and Tanzania and found compelling evidence for competition in mixed-strain infections: overall parasite density did not increase with additional strains, and densities of individual chloroquine-sensitive (CQS) and chloroquine-resistant (CQR) strains were reduced in the presence of competitors. We also found that CQR strains exhibited low densities compared with CQS strains (in the absence of chloroquine), which may underlie observed declines of chloroquine resistance in many countries following retirement of chloroquine as a first-line therapy. Our observations support a key role for within-host competition in the evolution of drug-resistant malaria. Malaria control and resistance-management efforts in high-transmission regions may be significantly aided or hindered by the effects of competition in mixed-strain infections. Consideration of within-host dynamics may spur development of novel strategies to minimize resistance while maximizing the benefits of control measures.

Results from regular drug efficacy monitoring should always be interpreted in the context of the many limitations inherent to attributing apparent treatment failures to antimalarial resistance, as concisely summarized by Hamed and Kuhen (1). Notably, in study settings where direct supervision of the evening doses of artemether-lumefantrine (AL) is operationally infeasible or culturally unacceptable, low efficacy can also potentially be attributed to inaccurate dosing or underdosing. Details of how we strived to guarantee participant compliance with evening doses in lieu of direct supervision are found in our original report (2). Importantly, procedures were identical in both provinces, and we have no indication that participant adherence to the evening doses or food consumption guidelines was different in Zaire Province, where we found lower efficacy of AL. While lack of direct observation of doses and nonadherence to guidelines regarding food consumption with drug administration have been shown to be associated with lower blood lumefantrine levels, they have not been directly associated with decreased efficacy of AL (3, 4). | | While in vitro susceptibility testing requires infrastructure rarely found at drug efficacy monitoring sites, there is a long history of complementing clinical outcome data with testing for known molecular markers of resistance (5). Samples from treatment failures with mutations associated with resistance provide more evidence of resistance than clinical outcome data alone. In our study, the detection of pfmdr1 haplotypes previously associated with decreased sensitivity to lumefantrine (6) and the absence of mutations associated with artemisinin resistance in AL treatment failures support the hypothesis of lumefantrine resistance. | | Notably, the NFD and NYD pfmdr1 haplotypes predominated not only in recrudescent infections but also in reinfections in the AL arms. Contrary to Hamed and Kuhen's assertion, reinfections observed during follow-up do provide important data on efficacy. For the calculation of uncorrected efficacy, an important outcome for therapeutic efficacy studies, both reinfections and recrudescences are considered treatment failures. The reporting and interpretation of uncorrected efficacy results are standard components of drug efficacy monitoring for two primary reasons (7,–9). First, uncorrected efficacy estimates are not subject to the limitations and potential biases of using genotyping data to differentiate reinfection from recrudescence. Second, uncorrected efficacy results provide data concerning the posttreatment prophylactic effect of the partner drug in artemisinin-based combination therapies and thus the proportion of clients that will require retreatment, data of increasing interest to malaria control programs. The reinfection rates in the AL arms in Zaire and Uíge Provinces provide a good example of the potential utility of reinfection rates. The reinfection rate in Zaire (13%) was measured as more than twice the rate in Uíge (5.1%). This was despite our screening data, where 67% of the children screened tested positive for malaria in Uíge, compared to 53% in Zaire, indicating that transmission was almost certainly higher in Uíge at the time of our study. This unexpectedly high rate of reinfection in Zaire could therefore point to a parasite population that is less sensitive to lumefantrine and that consequently has a higher likelihood of successfully invading patients with subtherapeutic doses of lumefantrine following AL treatment (10). | | Finally, we would like to reaffirm that while certain screened children had parasitemia levels above 100,000 parasites/μl, only children with parasitemia levels between 2,000 and 100,000 parasites/μl were enrolled in the study, as described in Materials and Methods. Given that no children with parasitemia levels above 100,000 parasites/μl were enrolled in any of the study arms at either site, differences in hyperparasitemia rates between the Uíge and Zaire AL arms cannot explain the difference in efficacy as Hamed and Kuhen suggest.

Routine therapeutic efficacy monitoring to measure the response to antimalarial treatment is a cornerstone of malaria control. To correctly measure drug efficacy, therapeutic efficacy studies require genotyping parasites from late treatment failures to differentiate between recrudescent infections and reinfections. However, there is a lack of statistical methods to systematically classify late treatment failures from genotyping data. A Bayesian algorithm was developed to estimate the posterior probability of a late treatment failure being the result of a recrudescent infection from microsatellite genotyping data. The algorithm was implemented using a Monte Carlo Markov Chain approach and was used to classify late treatment failures using published microsatellite data from therapeutic efficacy studies in Ethiopia and Angola.The algorithm classified 81% of the Ethiopian and 95% of the Angolan late treatment failures as either likely reinfection or likely recrudescence, defined as a posterior probability of recrudescence of <0.1 or >0.9, respectively. The adjusted efficacies calculated using the new algorithm differed from efficacies estimated using commonly-used methods for differentiating recrudescence from reinfection. In a high-transmission setting such as Angola, as few as fifteen samples needed to be genotyped in order to have enough power to correctly classify treatment failures. Analysis of microsatellite genotyping data for differentiating between recrudescence and reinfection benefits from an approach that both systematically classifies late treatment failures and estimates the uncertainty of these classifications. Researchers analyzing genotyping data from antimalarial therapeutic efficacy monitoring are urged to publish their raw genetic data and to estimate the uncertainty around their classification.

The development of resistance to antimalarials is a major challenge for global malaria control. Artemisinin-based combination therapies, the newest class of antimalarials, are used worldwide but there have been reports of artemisinin resistance in Southeast Asia. In February-May 2013, we conducted open-label, nonrandomized therapeutic efficacy studies of artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) in Zaire and Uige Provinces in northern Angola. The parasitological and clinical responses to treatment in children with uncomplicated P. falciparum monoinfection were measured over 28 days and the main outcome was PCR-corrected adequate clinical and parasitological response (ACPR) proportion on day 28. Parasites from treatment failures were analyzed for the presence of putative molecular markers of resistance to lumefantrine and artemisinins, including the recently identified mutations in the K13-propeller gene. In the 320 children finishing the study, 25 treatment failures were observed, 24 in the AL arms and one in the DP arm. The PCR-corrected ACPR proportion on day 28 for AL was 88% (95% CI: 78-95) in Zaire and 97% (91-100) in Uige. For DP, it was 100% (95-100) in Zaire, and 100% (96-100) in Uige. None of the treatment failures had molecular evidence of artemisinin resistance. In contrast, 91% of AL treatment failures had markers associated with lumefantrine resistance on day of failure. The absence of molecular markers for artemisinin resistance and the observed efficacies of both drug combinations suggest no evidence of artemisinin resistance in northern Angola. There is evidence of increased lumefantrine resistance in Zaire, which should continue to be monitored.