Recurrence of Plasmodium malariae and P. falciparum Following Treatment of Uncomplicated Malaria in North Sumatera With Dihydroartemisinin-Piperaquine or Artemether-Lumefantrine

Abstract

Background

We assessed the efficacy of artemisinin-based combination therapies for treatment of uncomplicated falciparum malaria, with or without co-infecting Plasmodium spp., in Sumatera, Indonesia.

Methods

Febrile patients aged >6 months with uncomplicated P. falciparum were randomized to receive dihydroartemisinin-piperaquine or artemether-lumefantrine, plus single-dose primaquine, and were followed for 42 days. Mixed Plasmodium infections were included; P. vivax infections received 14 days of primaquine. We retrospectively restricted the analysis to cases with polymerase chain reaction (PCR)–confirmed parasitemia. Recurrent parasitemia in follow-up was identified by species-specific nested PCR.

Results

Of the 3731 participants screened, 302 were enrolled and randomized. In the dihydroartemisinin-piperaquine arm, P. falciparum infections were confirmed by PCR in 59 participants, with mixed infections in 23 (39.0%). In the artemether-lumefantrine arm, P. falciparum infections were confirmed by PCR in 55 participants, with mixed infections in 16 (29.0%). Both regimens were well tolerated, and symptoms improved rapidly in all treated participants. In the dihydroartemisinin-piperaquine arm, 1 P. falciparum recurrence (on day 7) and 6 P. malariae recurrences (1 had a mixed infection with P. falciparum) were identified during days 3–42 of follow-up. In the artemether-lumefantrine arm, 1 P. falciparum/P. malariae/P. vivax recurrence occurred on day 35. Submicroscopic persistence occurred during follow-up in 21 (37%) of 57 receiving dihydroartemisinin-piperaquine and 20 (39%) of 51 receiving artemether-lumefantrine.

Conclusions

In Sumatera, both regimens effectively cleared initial parasitemia, but P. falciparum and P. malariae persisted in some individuals. Molecular species detection should be deployed in antimalarial efficacy trials in Indonesia.

Trial registration

NCT02325180.

Artemisinin-based combination therapy (ACT), co-formulating a short-acting artemisinin with a long-acting partner drug, is currently recommended for uncomplicated falciparum malaria. The widespread deployment of ACT, together with other control strategies, has reduced malaria morbidity and mortality, particularly in Africa [1]. ACT efficacy is threatened by Plasmodium falciparum with reduced susceptibility to both artemisinin and its partner drugs in the Greater Mekong subregion (GMS) [2–5]. Declining ACT efficacy against P. falciparum to ACT in this region raises concern that multidrug resistance may emerge in other malaria-endemic countries [6, 7].

Indonesia lies to the south and east of mainland Southeast Asia, and nearly 70 million of its population are at risk of malaria, including multispecies co-infections [8–10]. The ACT artesunate-amodiaquine, plus a single dose of primaquine to clear gametocytes, has been used for the treatment of uncomplicated falciparum malaria in Indonesia since 2004 [11]. Efficacy <90% was reported, and this combination was subsequently replaced by dihydroartemisinin-piperaquine (DP), plus a single dose of primaquine [12, 13]. For non-falciparum malaria, the standard 3-day course of DP is also recommended in combination with 1–14 days of primaquine for certain infecting species [14]. Published estimates of ACT efficacy against the non-falciparum parasite species in Indonesia exist only for P. vivax, which commonly occurs either alone or in co-infections with P. falciparum [8, 15], but no studies have adequately assessed the in vivo susceptibility of P. malariae or P. knowlesi, which co-circulate in Western Indonesia [8].

DP efficacy throughout Indonesia is reported to be excellent, and there are no reports of delayed clearance of P. falciparum, a sign of artemisinin resistance [13, 15–18]. However, a potential threat remains of artemisinin-resistant parasites being introduced from the GMS [6], adjacent to West Indonesia, or from Niugini (PNG), with a land border to East Indonesia [7]. Thus, there is a need to evaluate P. falciparum populations in Indonesia for susceptibility to current regimens in vivo. We aimed to compare the efficacy of DP, the first-line treatment for falciparum malaria, with artemether-lumefantrine (AL), also licensed in Indonesia, for treatment of uncomplicated falciparum malaria as single- and multispecies infections. The work was carried out in the North Sumatera province, which is a short sea crossing from Southern Thailand.

METHODS

Study Site and Participants

We conducted a prospective, open-label, randomized comparison of DP and AL for the treatment of uncomplicated falciparum malaria in the Batubara, Langkat, and South Nias regencies in the North Sumatera province, Western Indonesia (Supplementary Figure 1). Patients with uncomplicated P. falciparum malaria (including mixed infections with non-falciparum species) were eligible if they were aged >6 months, had fever/fever history (temperature ≥37.5°C) in the past 48 hours, and parasitemia >250 asexual parasites per µL, as determined by microscopic examination. Exclusion criteria were signs and symptoms of severe or complicated malaria, severe malnutrition, hemoglobin <7 g/dL, nonmalaria febrile illness, pregnancy or lactation, known hypersensitivity or allergy to study drugs, any antimalarial drug use in the previous 2 weeks, and any study drug use in the past 4 weeks.

The study was registered at ClinicalTrials.gov with the identifier NCT02325180 and approved by the Universitas Sumatera Utara (USU; August 2014; identifier 401/KOMET/FK USU/2014) and London School of Hygiene and Tropical Medicine (LSHTM; October 2014; Ref: 8504) research ethics committees. Consent forms were prepared in English and translated into Bahasa Indonesia. All adult participants and guardians of those <18 years old provided written informed consent.

Procedures

DP (Duo-Cotecxin, Holley Pharmaceutical, Hangzhou, Zhejiang Province, China; 40 mg dihydroartemisinin, 320 mg piperaquine) was given at doses of 2.25 mg/kg/dose and 18 mg/kg/dose, administered at enrollment, 24 hours, and 48 hours. AL (Coartem, Novartis Pharma, China; 40 mg artemether, 240 mg lumefantrine) was given according to weight, with 1 half-tablet for every 5 kg of body weight, at enrollment and hours 8, 24, 36, 48, and 60. DP was given on an empty stomach, and AL was given with biscuits or milk. Administration of all doses was supervised by study staff. Re-administration of a full dose or half-dose occurred if the patient vomited within 30 minutes or 30–60 minutes after treatment, respectively. As set out in Indonesian Government Treatment Guidelines, an additional single dose of primaquine at 0.75 mg/kg was given at enrollment for P. falciparum infection, and 14-day treatment with 0.25 mg/kg of primaquine per day was administered for all participants who were microscopy-positive for P. vivax at enrollment, commencing on day 42 [11].

Signs, symptoms, and adverse events were assessed and recorded before and during treatment and at all follow-up visits. Finger-prick blood samples provided blood films for microscopic assessment and dried filter paper blood spots for post hoc molecular analysis. Parasitological assessments were made from Giemsa-stained thick and thin blood smears and were single-read by a study team microscopist.

Parasite DNA was isolated from filter paper, and polymerase chain reaction (PCR) amplification of 18S rRNA genes for molecular identification of Plasmodium spp., pfmdr1 amplification for P. falciparum, and pksicavar amplification for P. knowlesi was performed as previously described [8]. Parasite clearance at days 0, 1, 2, and 3 was measured using microscopy and by quantitative PCR (qPCR) for amplification of genus-specific pgmet normalized to the human β-tubulin gene humtubb, as previously described [19, 20]. Hemoglobin levels were assessed using HemoCue at enrollment and day 28.

Recurrent P. falciparum infections, identified by microscopy during routine follow-up (days 1, 2, 3, 7, 14, 21, 28, 35, 42), were treated with quinine (10 mg of salt/kg given 3 times a day for 7 days) and doxycycline (100 mg twice a day for 7 days), except for children aged <8 years and pregnant women, who were treated with quinine and clindamycin. Patients who developed a non-falciparum Plasmodium infection were re-treated according to the national guidelines [11]. Recurrent infections identified by microscopy were confirmed post hoc by species-specific nested PCR amplification of 18S rRNA or merozoite antigen genes [8, 21]. Subpatent P. falciparum recurrences on days 28 and 42, or the latest available sample, were identified post hoc by PCR amplification of the pfmdr1 gene [22].

Outcomes

The primary end point defined in the study protocol was 42-day adequate clinical and parasitological response (ACPR) following DP and AL treatment for P. falciparum infection, as defined by the World Health Organization (WHO) [21]. Secondary and exploratory end points included qPCR parasite clearance over days 0 to 3; the proportion of patients with parasitemia at day 3 measured by microscopy and qPCR; efficacy of treatment against PCR-confirmed non-falciparum Plasmodium spp.; and the proportion of patients with subpatent Plasmodium spp. recurrences at days 28 and 42.

Statistical Analysis

A sample size of 150 participants in each group would provide 80% power to show the noninferiority of AL compared with DP at day 42, with an α of .05 and δ of 5%.

Data were double-entered and analyzed using Stata IC (version 15; StataCorp, TX, USA). Those lost to follow-up or who withdrew from the study were censored as having treatment failure. Only those outcomes determined in the PCR-defined population are presented in this report. Odds ratios and 95% confidence intervals were estimated for testing associations; P values of ≤.05 were considered statistically significant.

RESULTS

A total of 3731 participants were screened from January to June 2015 in the Regencies of in Batubara, Langkat, and South Nias (Supplementary Figure 1), as previously described [8]. Three hundred two individuals, comprising 263 P. falciparum infections and 39 mixed P. falciparum/P. vivax, as identified by microscopy, were eligible for enrollment and randomly assigned to receive DP (n = 153) or AL (n = 149) (Figure 1). Both regimens were well tolerated, and symptoms improved rapidly in all treated subjects. A single P. falciparum infection failed to clear by day 3 by microscopy in a patient receiving DP; post hoc PCR confirmed P. falciparum DNA (alone) at the day of enrollment. P. malariae and P. falciparum DNA were detected together at day 3 in this individual (Figure 1), and P. falciparum gametocytes were observed in the blood-film on days 1, 2, and 3, suggesting a chronic infection.

We performed species-specific nested PCR retrospectively on DNA from enrollment samples of all 302 randomized individuals. Surprisingly, Plasmodium spp. DNA was successfully detected in only 195 individuals (Figure 1), indicating that lack of accuracy in microscopic diagnosis had resulted in the inappropriate recruitment of individuals without a detectable malaria infection. Further, P. falciparum parasite DNA was specifically amplified in just 117 randomized patients (38.8%), in 40 of whom evidence was found of co-infecting Plasmodium spp. (Table 1). As a result of these findings, and excluding 5 individuals who did not complete 3 days of ACT treatment, we established a PCR-defined cohort (n = 190) and performed an exploratory efficacy assessment for each treatment group on a modified per-protocol basis, in which both enrollment and follow-up parasite detection was performed by PCR (Figure 1). Comparison of baseline characteristics for the 114 evaluable P. falciparum–infected individuals included in this analysis is given in Table 2.

Within the PCR-defined cohort, microscopically identified recurrent P. falciparum infections during follow-up were reported for 22 patients (17 for DP, 5 for AL). Species-specific PCR on the follow-up samples failed to detect parasite DNA in 10 and 4, respectively (Figure 1). Of the remainder, the majority harbored P. malariae alone (at days 7, 14, 14, 21, and 42) or mixed (day 3, 35), with only a single PCR-confirmed case of recurrent P. falciparum mono-infection (day 7). P. vivax DNA was detected in a single recurrence in the AL-treated group of the PCR-defined cohort at day 35, mixed with P. falciparum and P. malariae. Therefore, the specificity of microscopic identification of recurrent parasitemia was poor in our study, as judged by post hoc PCR.

An exploratory analysis of drug efficacy was then performed on both treatment arms of our PCR-defined cohort. First, we investigated parasite clearance dynamics of PCR-confirmed P. falciparum infection. We generated qPCR data for all enrolled study subjects for which DNA samples were available at days 0, 1, 2, and 3. Of the 114 confirmed P. falciparum patients, 60 were evaluated by qPCR at all 4 time points, 36 in the DP-treatment group and 24 in the AL group. Over 70% of patients in both groups cleared qPCR-detectable Plasmodium DNA within 24 hours, and only 4 (11%) and 3 (12%) receiving DP and AL, respectively, remained qPCR-positive at day 3 (Figure 2). Ninety percent or greater reduction in parasite density within 24 hours was achieved in 97% and 96% of patients treated with DP and AL, respectively. These data are consistent with continuing susceptibility to artemisinin in this P. falciparum population.

Second, with PCR-confirmed P. falciparum infections at enrollment, we identified parasite recurrence by nested PCR amplification of the pfmdr1 locus in all available 4- and 6-week follow-up samples. P. falciparum DNA was detected on days 28 or 42 in 41% of evaluable patients in the DP group (n = 59), and 35% in the AL group (n = 55; odds ratio, 0.833; 95% confidence interval, 0.365–1.89; P = .636) (Figure 3). Remarkably, all 24 patients from Langkat harbored PCR-detectable P. falciparum DNA at day 28 or day 42, whereas in Batubara and Nias the proportion was much lower, at 6/30 (20%) and 13/60 (22%), respectively.

Third, we explored ACT efficacy in patients harboring only non-falciparum malaria parasites, as measured by parasite clearance in the first 72 hours of treatment. Of 76 evaluable patients not harboring P. falciparum, 50 were PCR-positive for P. vivax at day 0, including 28 where this was the sole species (Table 1). Parasite clearance was measured in 30 of these individuals: All cleared qPCR-detectable parasitemia within 48 hours, except for 1 individual treated with DP who cleared parasitemia within 72 hours and 1 individual treated with AL who harbored persistent day 3 parasitemia, estimated as 0.3% of the starting parasite density. Three of 17 AL-treated patients with P. vivax (alone or mixed with P. knowlesi and/or P. malariae) were qPCR-positive at day 28 or day 42, equivalent to PCR-detected treatment failure of 17.6%, and 1 of 31 DP-treated patients with P. vivax (alone or mixed) was qPCR-positive at day 28, equivalent to a PCR-detected treatment failure of 3.2%. Of 19 P. knowlesi mono-infections, 8 generated parasite clearance data, of which 1 was qPCR-positive on day 3; this AL-treated individual was again qPCR-positive on day 28. Four individuals with P. malariae monoinfections at enrollment and 1 with both P. malariae and P. knowlesi were all qPCR-negative within 24 hours of commencing treatment with AL (2) or DP (3). Our qPCR detection target is specific only to the genus level, and further identification of the species present was unsuccessful in post-treatment isolates, which were of a low density. However, later recurrence of P. malariae did occur in 7 individuals originally treated for PCR-confirmed P. falciparum. In conclusion, this off-protocol analysis demonstrates the potential for qPCR to provide estimates of post-treatment parasite clearance dynamics and to detect recrudescence in non-falciparum malaria.

Discussion

We report the results of an open-label efficacy comparison of DP (n = 153) vs AL (n = 149) for the treatment of uncomplicated malaria in 3 Regencies of the North Sumatera province, where P. falciparum co-circulates with P. vivax, P. knowlesi, and P. malariae. This is the Indonesian region closest to the GMS, where P. falciparum with reduced susceptibility to artemisinin, associated with slow clearance of ACT-treated infections, first emerged. We sought information on the current efficacy of ACT in these settings. Poor diagnostic accuracy of clinic microscopy led to enrollment and randomization of many individuals who did not meet the enrollment criteria, thus hampering any meaningful analysis on an intention-to-treat basis. Similarly, post hoc species-specific PCR indicated that many microscopically identified recurrent infections in follow-up did not harbor Plasmodium spp. DNA and had been misclassified. We therefore deviated from our original study protocol and restricted analysis to a PCR-defined per-protocol cohort comprising 102 and 88 PCR-confirmed infections and assessed drug efficacy using both qualitative and quantitative DNA detection end points.

Clearance of PCR-confirmed P. falciparum infections over the first 72 hours of treatment (measured by qPCR for 62 individuals) was rapid for both regimens, with only 11% and 12% remaining qPCR-positive at day 3. This suggests that P. falciparum in Western Indonesia retains susceptibility to artemisinin, although the occurrence of 1 DP-treated individual with microscopy-positive (PCR-confirmed) early treatment failure on day 3 is of concern. Genotyping of this recrudescent day 3 infection at resistance-associated loci has been reported elsewhere [22]; it was found to carry a wild-type pfkelch13 gene, pfcrt 72–76 haplotype SVMNT, pfmdr1 86/184/1246 haplotype YYD, and multiple copies of the pfmdr1 gene. The pfcrt genotype is known to be an important modulator of parasite response to a variety of current antimalarials, including artemisinin, lumefantrine, and piperaquine [23, 24], but no studies have systematically examined the impact of the SVMNT haplotype on P. falciparum susceptibility to these drugs. We recently identified isolates from the China-Myanmar border region with the SVMNT haplotype of pfcrt also harboring pfk13 propeller-domain mutations [25].

Two elements of our study provide reasons to be cautious regarding any firm conclusions about P. falciparum artemisinin susceptibility. First, our study was planned as a treatment trial for falciparum malaria patients, with and without other co-infecting species, expected to be P. vivax (https://clinicaltrials.gov: NCT02325180), but randomization between the 2 treatment arms was seriously compromised because of the large number of uninfected enrollees (Figure 1, Table 1). Randomization may also have potentially been compromised by the ad hoc screening procedure, in which participants were identified by different means across the 3 sites [8]. For this reason, we do not present any comparative statistical analysis between the drug arms, but present descriptive statistics only. Second, as demonstrated by our community-wide study, carried out simultaneously during recruitment, there is a complex mix of 4 Plasmodium species circulating in our study areas [8]. The use of DNA amplification methods to confirm the species present and to verify cases of putative treatment failure, was therefore important in determining the real (per-protocol) denominator of treatment failure in each treatment group, but is not a recognized approach to therapeutic efficacy studies.

A minority of microscopically detected parasite recurrences were confirmed to be PCR-positive for Plasmodium spp., and surprisingly the majority of these were due to P. malariae, despite P. vivax (68 individuals) and P. knowlesi (65 individuals) being much more prevalent at enrollment (Table 1). Our results therefore suggest that artemisinin is effective at rapidly clearing the majority of P. vivax and P. knowlesi infections and that P. malariae can rebound to microscopy- or PCR-detectable levels 2–6 weeks after ACT treatment. This is consistent with reports of P. malariae recurrence after ACT treatment in Ghana and Uganda [26, 27], with suggestions that the 72-hour intra-erythrocytic cycle of P. malariae provides an opportunity for this species to survive 3-day artemisinin exposure [28, 29]. This supports the argument that extended artemisinin regimens may be required for radical cure [30, 31], but these findings must be interpreted cautiously, given the problems with parasite identification at screening and enrollment.

Estimates of drug efficacy against PCR-confirmed P. falciparum infection were determined in each group at days 28 and 42. Here we found evidence of submicroscopic parasite persistence at day 28 and/or day 42 in 41% and 36% of the DP and AL groups, respectively (Figure 3). We conclude that, although both regimens seem effective at clearing initial parasitemia by qPCR, P. falciparum recurrence was a common occurrence with both regimens. Unfortunately, these presumably subpatent infections were not of sufficient parasite density to permit strain genotyping, but it is safe to say that neither 3-day ACT regimen provided protection against persistent submicroscopic P. falciparum for treated individuals at 4–6 weeks. As previously argued, longer sequential ACT treatment regimens over 6 days may provide both effective initial parasite clearance and prevention of P. falciparum recurrence [30], a strategy also likely to prove beneficial against P. malariae in North Sumatera [31].

Our results present a confusing picture of apparent good artemisinin efficacy, evidenced by satisfactory parasite clearance of all infecting Plasmodium species by day 3, overlaid by complex patterns of multispecies parasite recurrence manifested as PCR-detectable P. falciparum and microscopy-detectable P. malariae. Parasite DNA detection is not a recognized measure of antimalarial treatment outcome, and only microscopically detected parasitological treatment failure is recognized by current WHO protocols. This remains the appropriate current standard, as there are uncertainties as to the exact meaning of persistent parasite DNA in the treated individual. For example, in the case of P. falciparum, gametocyte emergence occurs late in infection and can provide a DNA biomarker in a treated individual despite effective clearance of all replicating asexual parasites [20]. It can also be argued that drug-killed parasites bequeath sufficient circulating DNA in the host that a positive PCR result does not necessarily mean viable, replicating parasites are still present. However, the lack of evidence that P. vivax DNA persisted in our treated individuals and work in both rodent malaria [32] and ACT-treated human infections where even hyperparasitemic patients rapidly become PCR-negative [19] strongly suggest this is not the case. Nevertheless, the results presented here suggest that work toward a new protocol for therapeutic efficacy studies for antimalarial drugs, in which molecular detection methods are intrinsic to enrollment as well as to outcomes, is needed.

The complexity of malaria in our study areas may be the result of human communities being isolated with poor access to diagnosis and treatment for malaria, as well as a tendency for low-density, multispecies chronic infections to develop among individuals exposed to regular mosquito challenge in these settings [8, 26]. Such established infections elicit immune responses from the host and can enter a steady state maintained by antigenic variation, and therefore may be more difficult to treat than acute infections [27]. This provides an additional rationale for the evaluation of extended ACT regimes as a means to reduce persistent parasitemia in treated individuals [30]. The complexity of malaria infections in Sumatera is a challenge for point-of-care diagnosis as well as for patient recruitment for antimalarial efficacy trials, as recently noted in neighboring Aceh Province [10], suggesting that further diagnostic and surveillance capacities are needed in the region [33].

Supplementary Data

Supplementary materials are available at Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Acknowledgments

We thank the participants, their families, and the community health workers and village leaders who gave their cooperation and support to the work and Gisela Henriques for her contribution to the field studies in South Nias. We are grateful to staff and students of the Universitas Sumatera Utara, Medan, for practical assistance and logistical support.

Author contributions. Inke Lubis: study design, protocol writing, data collection, supervision of field staff, laboratory investigations, data analysis, data interpretation, writing of manuscript, figures. Hendri Wijaya: data collection, supervision of field staff, laboratory investigations, revision of manuscript. Munar Lubis: study design, data collection, supervision of field staff, laboratory investigations, revision of manuscript. Chairuddin Lubis: study design, data collection, supervision of field staff, laboratory investigations, revision of manuscript. Khalid Beshir: study design, data collection, laboratory investigations, data analysis, data interpretation, revision of manuscript. Sarah Staedke: study design, protocol writing, writing of manuscript. Colin Sutherland: study design, protocol writing, supervision of laboratory, laboratory investigations, data analysis, data interpretation, writing of manuscript, figures, manuscript submission. The corresponding author confirms that he had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Disclaimer. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The principal investigator (I.N.D.L.) had full access to all the data in the study; the corresponding author (C.J.S.) had full access to all the data in the study and had final responsibility for the decision to submit this manuscript for publication.

Financial support. This study was funded by a PhD scholarship from the Directorate General of Higher Education of Indonesia to I.N.D.L. C.J.S. is supported by Public Health England.

Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Prior presentation. Some of the information in the paper was presented at the Evolving Artemisinin Resistance Workshop at Georgetown University, Washington, DC, in November 2019.

References