• View in gallery

    (A) qPCR-determined parasite prevalence for artemether–lumefantrine (AL) and pyronaridine–artesunate (PA), shown as the proportion of positive participants, by follow-up day. Error bars indicate 95% confidence intervals (CIs). (B) Direct-on-blood PCR nucleic acid lateral flow immunoassay (db-PCR-NALFIA)–determined parasite prevalence for AL and PA, shown as the proportion of positive participants, by follow-up day. Error bars indicate 95% CIs.

  • View in gallery

    Median qPCR-determined parasite density, as a percentage of the enrollment value, for artemether–lumefantrine (AL) (solid line) and pyronaridine–artesunate (PA) (dashed line). PCT95 indicates time to 95% reduction in parasite density.

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Molecular Detection of Residual Parasitemia after Pyronaridine–Artesunate or Artemether–Lumefantrine Treatment of Uncomplicated Plasmodium falciparum Malaria in Kenyan Children

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  • 1 Department of Medical Microbiology, Academic Medical Center, Amsterdam, The Netherlands;
  • 2 Human Health Division, International Centre of Insect Physiology and Ecology, Mbita Point, Kenya

Artemisinin resistance is rapidly rising in Southeast Asia and may spread to African countries, where efficacy estimates are currently still excellent. Extensive monitoring of parasite clearance dynamics after treatment is needed to determine whether responsiveness to artemisinin-based combination therapies (ACT) is changing in Africa. In this study, Kenyan children with uncomplicated falciparum malaria were randomly assigned to pyronaridine–artesunate (PA) or artemether–lumefantrine (AL) treatment. Parasite clearance was evaluated over 7 days following the start of treatment by quantitative polymerase chain reaction (qPCR) and direct-on-blood PCR nucleic acid lateral flow immunoassay (db-PCR-NALFIA), a simplified molecular malaria diagnostic. Residual parasitemia at day 7 was detected by qPCR in 37.1% (26/70) of AL-treated children and in 46.1% (35/76) of PA-treated participants (P = 0.275). Direct-on-blood PCR nucleic acid lateral flow immunoassay detected residual parasites at day 7 in 33.3% (23/69) and 30.3% (23/76) of AL and PA-treated participants, respectively (P = 0.692). qPCR-determined parasitemia at day 7 was associated with increased prevalence and density of gametocytes at baseline (P = 0.014 and P = 0.003, for prevalence and density, respectively) and during follow-up (P = 0.007 and P = 0.011, respectively, at day 7). A positive db-PCR-NALFIA outcome at day 7 was associated with treatment failure (odds ratio [OR]: 3.410, 95% confidence interval [CI]: 1.513–7.689, P = 0.003), but this association was not found for qPCR (OR: 0.701, 95% CI: 0.312–1.578, P = 0.391). Both qPCR and db-PCR-NALFIA detected substantial residual submicroscopic parasitemia after microscopically successful PA and AL treatment and can be useful tools to monitor parasite clearance. To predict treatment outcome, db-PCR-NALFIA may be more suitable than qPCR.

INTRODUCTION

Artemisinin-based combination therapies (ACTs) are first-line treatment of uncomplicated Plasmodium falciparum malaria in many African countries, where they show excellent cure rates despite concerns about increasing resistance in Southeast Asia.1,2 Artemisinin resistance is characterized by delayed parasite clearance and associated with point mutations in the P. falciparum kelch protein gene on chromosome 13 (K13).1,3 Even though the polymorphisms related to P. falciparum artemisinin resistance in Southeast Asia are not prevalent in sub-Saharan Africa, several (other) K13 coding polymorphisms circulate in African countries.47 The impact of these K13 mutations present in Africa on artemisinin resistance is unknown.8 However, an increase in parasite clearance time after artemether–lumefantrine (AL) treatment was recently reported in coastal Kenya.9 Submicroscopic parasitemia after ACT treatment has been described in Sudan and Uganda.1012 In western Kenya, residual submicroscopic parasitemia was found to be common after ACT treatment and associated with increased gametocyte carriage, higher transmission potential to mosquitos, and an increased likelihood of recurrent parasitemia on day 28 or 42.13 Extensive monitoring of parasite clearance dynamics after treatment is thus needed to determine whether responsiveness to ACT is changing.

To ensure the availability of effective antimalarials, also in areas of drug resistance, the development and evaluation of novel drugs is ongoing. An alternative to currently used first-line ACTs is pyronaridine–artesunate (PA) treament, which was found to be well tolerated and efficacious for the treatment of P. falciparum malaria and the blood stage of Plasmodium vivax malaria.14 Insight in microscopic and submicroscopic parasite clearance after PA treatment is important not only to investigate current clearance rates in relation to other ACTs, but also to facilitate potential future comparisons between pre- and postimplementation clearance estimates.

To perform extensive follow-up of patients after treatment, sensitive laboratory techniques are becoming increasingly important. Microscopy is cheap, quantitative, and allows species determination but has a relatively high limit of detection (LoD): about 20 parasites/μL for an expert microscopist and ∼100 parasites/μL under field conditions.15 Low parasitemia is common shortly after treatment and might go undetected when using microscopy.13 Antigen-based rapid diagnostic tests (RDTs) are fast and easy to perform but have limited sensitivity as well (LoD: ∼100 parasites/μL).16 In addition, histidine-rich protein II–based RDTs are unsuitable for follow-up because circulating antigens persist in the circulation up to 1 month after treatment and therefore cannot distinguish between present and past infection during this period.17

The availability of a sensitive and reliable method to monitor treatment efficacy would help clinicians and researchers to detect possible resistance early and adjust treatment regimens when and where necessary. Molecular tools may be suitable for this purpose, especially when they are able to predict a failure shortly after treatment initiation. For example, quantitative nucleic acid sequence–based amplification (QT-NASBA) on day 7 after treatment could correctly predict late clinical or parasitological failure (LCF/LPF) in most cases.18 Furthermore, detection of residual parasitemia at day 3 after treatment by qPCR was found to be associated with an increased risk of LCF/LPF, even for recurrent parasitemia classified as a new infection.13 However, molecular diagnostics are often difficult to implement in low-resource settings.19 Direct-on-blood PCR nucleic acid lateral flow immunoassay (db-PCR-NALFIA) is a sensitive molecular tool that has several implementation advantages over QT-NASBA and qPCR in the sense that it is fast, does not require nucleic acid extraction, and has an easy lateral flow–based read-out system.20,21 These characteristics make db-PCR-NALFIA a potentially suitable tool to monitor treatment efficacy in resource-restricted settings.

The aim of this study was to evaluate the use of qPCR and db-PCR-NALFIA to determine parasite clearance after treatment of uncomplicated falciparum malaria with PA or AL. Furthermore, the association between molecular test results on day 7 after treatment initiation and subsequent treatment failure or success was established for both qPCR and db-PCR-NALFIA.

MATERIALS AND METHODS

Study site and design.

This observational study was part of a phase III randomized clinical trial investigating the efficacy and safety of PA compared with AL in Kenyan children with uncomplicated P. falciparum malaria. Full efficacy and safety results from this trial will be published separately. The study was approved by the Ethical Review Committee of the Kenya Medical Research Institute (NON-SSC no. 479, registered at clinicaltrials.gov under NCT02411994). The main trial comprised 197 participants (101 received PA and 96 AL) and the present study included a random consecutive subset of 151 participants. Before randomization to PA or AL, written informed consent was obtained from a parent/guardian, after they had been fully informed about the purpose and logistics of the study, all their questions had been answered, and they agreed to participate.

Patients aged 6 months to 12 years, presenting with symptoms of uncomplicated malaria to the St. Jude’s Clinic, Mbita, were enrolled if they met the following inclusion criteria: having uncomplicated P. falciparum mono-infection with a parasitemia between 1,000 and 200,000 parasites/μL. Exclusion criteria were as follows: a hemoglobin level of < 6 g/dL, not being available for follow-up, living more than 10 km from the study clinic, use of antimalarial therapy in the previous 2 weeks, history of hepatic and/or renal impairment, severe malnutrition, known hypersensitivity to artemisinins, previous participation in this study, and current participation in other antimalarial drug intervention studies.

Following diagnosis, recruitment and randomization at day 0, a weight-dependent dose of either AL or PA was administered by pharmacy staff aware of treatment allocation. Clinical and other research staff were blinded to treatment allocation. Participants in the AL group received their morning dose at the research clinic under direct observation and parents were provided with the evening dose and local fatty food to administer at home. Pyronaridine–artesunate had to be administered only once per day and this was carried out at the research clinic under direct observation. Study medication was administered with fatty food or milk to all study participants. Treatment follow-up was carried out at the research clinic at days 0, 1, 2, 3, 7, 14, 28, and 42, according to the World Health Organization (WHO) recommendations.22 Children developing danger signs of severe malaria were immediately withdrawn and received rescue treatment or were referred to a nearby district hospital. Response to treatment (as determined by microscopy) was recorded using WHO definitions as early treatment failure, LCF, LPF, or adequate clinical and parasitological response (ACPR).22

Sample collection.

Capillary blood samples were collected by finger prick at day 0 and all follow-up days. For db-PCR-NALFIA, 100 μL was collected in an ethylenediaminetetraacetic acid tube (Sarstedt, Nümbrecht, Germany). An additional 100 μL was collected on Whatman 903 protein saver cards (GE Healthcare, Chicago, IL) for DNA extraction and conduction of qPCR and QT-NASBA (for gametocyte detection) in Amsterdam, the Netherlands. Filter papers were air-dried for 24 hours before being individually packed with silica and stored at −20°C. Microscopy slides were prepared directly from the finger prick. All test operators were blinded to treatment allocation and other test results.

Laboratory analyses.

Giemsa-stained thick- and thin-blood smears were prepared according to WHO guidelines and read by two local expert microscopists.23 A slide was considered negative when 100 high-power fields were examined in the thick smear at ×1,000 magnification and no parasites were observed. Parasitemia was determined from thick smears by counting the number of parasites against 200 leukocytes, with the assumption of 8,000 leukocytes/μL blood. When the number of parasites after counting 200 leukocytes was < 100, counting continued up to 500 leukocytes. Species determination was carried out using the thin smears.

To discriminate between new and recrudescent infections, genotyping was carried out using merozoite surface protein 1 and 2 and glutamate rich protein markers, according to WHO recommendations.24,25

The db-PCR-NALFIA assay can discriminate between pan-Plasmodium (P. falciparum, P. vivax, Plasmodium malariae, and Plasmodium ovale) and P. falciparum and was performed at St. Jude’s Clinic as described elsewhere.21 No nucleic acid extraction is required for this PCR and the readout was carried out on a lateral flow device.20

Nucleic acids were extracted from filter papers by Nuclisens easyMag (bioMérieux, Marcy-l’Étoile, France) and subsequently stored at −70°C. 18S P. falciparum qPCRs were performed on a CFX96 detection system (BioRad, Hercules, CA) as previously described.2628 Standard curves of P. falciparum 3D7 culture (104–100 parasites/μL) and negative controls (uninfected erythrocytes and water) were included in every run in duplicate. Quantification was performed according to the standard curve. Results were analyzed using CFX manager software (BioRad), with a set threshold of 100 relative fluorescence units. Samples were considered positive if they crossed this threshold within 40 PCR cycles, which corresponds to a parasite count of approximately 0.2/μL.

Gametocyte detection by QT-NASBA was performed as previously described,29 with minor modifications. The reaction mixture (5 μL) and sample (2.5 μL) were incubated for 2 minutes at 65°C and 2 minutes at 41°C. Enzyme was added (2.5 μL) and the reaction was allowed to run for 30 minutes at 41°C. Quantification was performed using standard curves of 103 to 10−1 gametocytes/μL.

Outcomes.

The primary aim of this study was to evaluate qPCR-estimated parasite clearance after PA treatment compared with AL treatment. Second, parasite clearance was also assessed by db-PCR-NALFIA, the parasite reduction ratio (PRR) 48 hours after treatment initiation (PRR48) was determined and the time to 95% reduction in parasite density (PCT95) was established. In addition, the association between the molecular test results at day 7 (qPCR or db-PCR-NALFIA) and treatment success or failure during follow-up was assessed. Finally, an exploratory analysis was carried out to evaluate differences in baseline characteristics and QT-NASBA-based gametocyte prevalence and density among participants with and without residual parasitemia at day 7.

Statistical analysis.

Stata software version 14.0 was used for statistical analyses (StataCorp, College Station, TX). Parasite clearance was evaluated by assessing parasite prevalence (qPCR and db-PCR-NALFIA) and relative density (qPCR) at day 0, 1, 2, 3, and 7 after initiation of treatment. The PRR48 was estimated as described previously,30,31 using qPCR. For participants negative by qPCR 48 hours after the start of treatment, the PRR48 was arbitrarily set at 105. The PCT95 was established using a log-linear model as described previously.30 The association between the molecular test result at day 7 (qPCR or db-PCR-NALFIA) and treatment success or failure during follow-up was assessed using logistic regression models. Differences between groups in parasite density were tested with the Wilcoxon rank sum test. Prevalence differences were tested with the χ2 or Fisher’s exact test.

RESULTS

Study population.

Parasite clearance was evaluated using qPCR and db-PCR-NALFIA in a consecutive subset of 151 participants from the main efficacy trial. From this subset, 73 received AL and 78 received PA (Table 1). For 130 of the 151 participants a complete dataset was available to estimate the association between test result at day 7 and treatment outcome during follow-up.

Table 1

Parasite prevalence and density estimates by follow-up day using qPCR and db-PCR-NALFIA

Artemether–lumefantrinePyronaridine–artesunateP value
qPCR
 Prevalence, % (n/N)
  Day 0100 (73/73)98.7 (77/78)0.332
  Day 197.2 (69/71)96.1 (74/77)0.717
  Day 276.8 (53/69)94.8 (73/77)0.002
  Day 362.3 (43/69)78.4 (58/74)0.035
  Day 737.1 (26/70)46.1 (35/76)0.275
 Density, % of enrollment value, median (IQR)
  Day 0100100N/A
  Day 11.746 (0.707–5.523)1.824 (0.677–5.023)0.8526
  Day 20.076 (0.026–0.453)0.159 (0.049–0.409)0.1144
  Day 30.031 (0.010–0.130)0.050 (0.014–0.242)0.2088
  Day 70.019 (0.002–0.122)0.021 (0.006–0.254)0.3976
db-PCR-NALFIA
 Prevalence, % (n/N)
  Day 0100 (73/73)100 (78/78)N/A
  Day 198.6 (69/70)97.4 (75/77)0.617
  Day 276.8 (53/69)80.3 (61/76)0.613
  Day 346.4 (32/69)62.2 (46/74)0.058
  Day 733.3 (23/69)30.3 (23/76)0.692

db-PCR-NALFIA = direct-on-blood PCR nucleic acid lateral flow immunoassay; N/A = not applicable. Density is reported as a percentage of the enrollment value (median [IQR]). P values represent between group differences. Prevalence differences were tested with the χ2 or Fisher’s Exact test. Differences in density were tested with the Wilcoxon rank sum test.

Residual parasitemia after treatment.

Based on microscopic clearance assessment of all participants in the main efficacy trial, both AL and PA were found to be effective with only two participants in the AL group (2.08%) and three in the PA group (2.97%) harboring parasites at day 3. One participant in the PA group had microscopically detectable asexual parasites at day 7 and received rescue treatment.

As expected, both molecular methods found higher prevalences of residual parasites during follow-up than microscopy. Prevalence determined by qPCR at day 0, 1, 2, 3, and 7 is shown in Figure 1A. At day 2, significantly more participants in the PA group were positive (94.8%), compared with the AL group (P = 0.002). Residual parasitemia at day 3 was observed in 62.3% and 78.4% of participants in the AL and the PA group, respectively (P = 0.035). By day 7, qPCR was still positive in 37.1% of participants in the AL group and in 46.1% in the PA group (P = 0.275) (Table 1). The parasite prevalence during follow-up as determined by db-PCR-NALFIA is shown in Figure 1B. By contrast to qPCR, no difference between treatment groups was found at day 2. Db-PCR-NALFIA estimates of residual parasitemia were overall slightly lower than qPCR estimates. At day 3, residual parasitemia was observed in 46.4% and 62.2% of participants in the AL and the PA group, respectively (P = 0.058). By day 7, db-PCR-NALFIA was positive in 33.3% and 30.3% of participants in the AL and the PA group, respectively (P = 0.692) (Table 1).

Figure 1.
Figure 1.

(A) qPCR-determined parasite prevalence for artemether–lumefantrine (AL) and pyronaridine–artesunate (PA), shown as the proportion of positive participants, by follow-up day. Error bars indicate 95% confidence intervals (CIs). (B) Direct-on-blood PCR nucleic acid lateral flow immunoassay (db-PCR-NALFIA)–determined parasite prevalence for AL and PA, shown as the proportion of positive participants, by follow-up day. Error bars indicate 95% CIs.

Citation: The American Journal of Tropical Medicine and Hygiene 99, 4; 10.4269/ajtmh.18-0233

The relative median parasite density during follow-up as determined by qPCR is shown in Figure 2 and was not found to be associated with treatment group (Table 1). At day 1, the parasite density was reduced by 98.3% in the AL group and 98.2% in the PA group (P = 0.853). The PCT95 was 17.2 hours for AL (interquartile range [IQR]: 14.2–22.3) and 17.4 hours for PA (IQR: 14.4–24.7), but these estimates need to be interpreted with caution because there were no measurements between baseline and day 1 and minor differences in time have a large impact on estimated density during the first 48 hours after treatment (Figure 2). By day 7, the density decreased to a median of 0.019% of the enrollment value (IQR: 0.002–0.122) in the AL group and to 0.021% (IQR: 0.006–0.254) in the PA group (P = 0.398) (Table 1).

Figure 2.
Figure 2.

Median qPCR-determined parasite density, as a percentage of the enrollment value, for artemether–lumefantrine (AL) (solid line) and pyronaridine–artesunate (PA) (dashed line). PCT95 indicates time to 95% reduction in parasite density.

Citation: The American Journal of Tropical Medicine and Hygiene 99, 4; 10.4269/ajtmh.18-0233

The median PRR48 was significantly higher in the AL group (2,315.4, IQR: 549.89–12,984) compared with the PA group (713.61, IQR: 249.26–2,615.3) (P = 0.001). However, no association was found between the PRR48 and residual parasitemia at day 7 as detected by qPCR for both treatment groups (Table 2). Participants with qPCR-detected parasites at day 7 had a higher parasite density at enrollment (P = 0.043, Table 3). No association was found between qPCR positivity at day 7 and treatment arm, age, hemoglobin concentration at baseline, or fever status at baseline.

Table 2

Parasite reduction ratio 48 hours after the start of treatment with either AL or PA

Artemether–lumefantrinePyronaridine–artesunateP value
PRR482,315.4 (549.89–12,984)713.61 (249.26–2,615.3)0.001
qPCR positive day 7qPCR negative day 7qPCR positive day 7qPCR negative day 7
PRR48, AL arm2,236.1 (476.44–8,014.7)2,930.4 (556.12–100,000)0.490
PRR48, PA arm526.23 (146.36–1,527.0)844.09 (287.53–2,801.6)0.140

AL = artemether–lumefantrine; PA = pyronaridine–artesunate; PRR48 = parasite reduction ratio 48 hours. Data are median (IQR). The PRR48 was estimated using qPCR as described previously.30,31 Differences in PRR48 were tested with the Wilcoxon rank sum test.

Table 3

Baseline characteristics of study participants with cleared or residual parasitemia at day 7 after treatment initiation with artemether–lumefantrine or PA, as determined by qPCR

qPCR treatment outcome on day 7P value
Positive (N = 61)Negative (N = 85)
Treatment arm PA, % (n/N)57.4 (35/61)48.2 (41/85)0.357
Age, years, median (IQR)6.7 (4–9.8)7 (4.4–9.8)0.657
Fever (axillary temperature > 37.5°C), % (n/N)47.5 (29/61)57.6 (49/85)0.227
Hemoglobin level, g/dL, mean (95% CI)12.0 (11.5–12.6)11.9 (11.5–12.3)0.632
Parasite density, qPCR, geometric mean (95% CI)9,467 (5,097–17,586)6,238 (4,222–9,215)0.043
Gametocyte prevalence, microscopy, % (n/N)4.92 (3/61)1.18 (1/85)0.172

CI = confidence interval; PA = pyronaridine–artesunate.

The microscopy-based gametocyte prevalence at baseline was low and not significantly higher in participants who were qPCR positive at day 7 (P = 0.172) (Table 3). Quantitative nucleic acid sequence–based amplification estimated gametocytemia, on the other hand, was common after treatment and gametocyte prevalence at enrollment, day 3 and day 7 was significantly higher in participants who were qPCR positive at day 7 compared with those who were not. Gametocyte density was also higher in participants who were qPCR positive at day 7, and this difference was statistically significant at day 0 and 7 but not at day 3 (Table 4). Participants gametocyte positive (by QT-NASBA) at day 0 had significantly higher day 0 parasitemia as estimated by qPCR compared with those who were QT-NASBA negative (P < 0.001, Wilcoxon rank sum test). This was not the case for participants gametocyte positive at day 3 nor for those positive at day 7.

Table 4

Quantitative nucleic acid sequence–based amplification detection of gametocytes in patients with qPCR-determined cleared or residual parasitemia at day 7

qPCR treatment outcome on day 7P value
Positive (N = 61)Negative (N = 85)
Gametocyte prevalence, % (n/N)
 Day 0100 (61/61)90.6 (77/85)0.014
 Day 343.3 (26/60)25.6 (21/82)0.027
 Day 727.9 (17/61)10.6 (9/85)0.007
Gametocyte density, geometric mean (95% CI)
 Day 08.616 (4.605–16.12)2.374 (1.425–3.956)0.003
 Day 34.182 (1.354–12.92)1.727 (0.633–4.710)0.380
 Day 710.43 (2.690–40.46)0.484 (0.112–2.101)0.011

CI = confidence interval.

Residual parasitemia at day 7 and recurrence during follow-up.

For 130 participants with qPCR and db-PCR-NALFIA–described clearance dynamics, the treatment outcome at day 28 and 42 was known: 34 had recurrent parasitemia detected by microscopy and 96 had an ACPR.

A positive qPCR result at day 7 was not significantly associated with parasite recurrence at day 28 or 42 (odds ratio [OR]: 0.701, 95% confidence interval [CI]: 0.312–1.578, P = 0.391). A positive db-PCR-NALFIA at day 7, on the other hand, was associated with recurrent parasitemia (OR: 3.410, 95% CI: 1.513–7.689, P = 0.003). This association was not confounded by age or enrollment parasite density. Treatment group AL may be weakly associated with increased parasite recurrence (OR of PA to AL: 0.531, 95% CI: 0.241–1.169, P = 0.116), but the association between db-PCR-NALFIA–detected residual parasitemia at day 7 and recurrence during follow-up remained significant after adjusting for treatment (OR: 3.326, 95% CI: 1.464–7.556, P = 0.004). Although the OR is valuable for research purposes, predictive values are more useful in clinical practice. Of the participants who were db-PCR-NALFIA positive at day 7, 19 had a recurrence and 26 an ACPR during follow-up, resulting in a low positive predictive value (42.2%, 95% CI: 31.9–53.3) for recurrent parasitemia. The negative predictive value, on the other hand, was 82.4% (95% CI: 75.8–87.4): of the 85 participants who were db-PCR-NALFIA negative at day 7, 70 had an ACPR. Thus, in most cases, a negative db-PCR-NALFIA test result at day 7 indicated an ACPR during follow-up. It should be noted that predictive values depend on the prevalence and may, thus, differ between settings.

For all 34 participants with recurrent parasitemia, genotyping was performed to make a distinction between recrudescent and new infections. Based on WHO criteria, six participants had a recrudescence, 25 had a new infection and three genotyping results were indeterminate. Residual parasitemia at day 7 as detected by qPCR remained unassociated with both new infections (OR: 0.713, 95% CI: 0.288–1.762, P = 0.463) and recrudescences (OR: 1.373, 95% CI: 0.266–7.079, P = 0.705). A positive db-PCR-NALFIA at day 7 was also not significantly associated with new infections (OR: 2.019, 95% CI: 0.830–4.913, P = 0.121) (Table 5) but db-PCR-NALFIA was positive at day 7 for all six participants with a recrudescence during follow-up. Importantly, although this finding suggests prediction of recrudescent infections, its clinical use will be limited because of the low positive predictive value. A negative db-PCR-NALFIA result at day 7, on the other hand, might be a useful indicator of ACPR.

Table 5

Association between db-PCR-NALFIA or qPCR test result at day 7 and treatment failure

db-PCR-NALFIAqPCR
OR (95% CI)P valueOR (95% CI)P value
Recurrence3.410 (1.513–7.689)0.0030.701 (0.312–1.578)0.391
RecrudescenceN/A*N/A*1.373 (0.266–7.079)0.705
Reinfection2.019 (0.830–4.913)0.1210.713 (0.288–1.762)0.463

CI = confidence interval; db-PCR-NALFIA = direct-on-blood PCR nucleic acid lateral flow immunoassay; N/A = not applicable; OR = odds ratio. Three indeterminate genotyping results were included in the recurrence but not in the recrudescence and reinfection analyses.

The OR could not be determined. All six recrudescent infections were positive by db-PCR-NALFIA at day 7.

DISCUSSION

This study showed substantial submicroscopic residual parasitemia after treatment with either PA or AL, despite good clearance estimates by microscopy. Residual parasitemia was detected by both qPCR and db-PCR-NALFIA, although prevalence estimates were slightly higher for qPCR. The db-PCR-NALFIA but not qPCR result at day 7 was found to be associated with treatment outcome during follow-up.

Submicroscopic parasitemia after ACT treatment has been described previously in the same study area, where one-third of ACT-treated study participants were found to carry submicroscopic parasites at day 3.13 In the present study, prevalence estimates were even higher: approximately two-thirds of study participants had qPCR-detected parasitemia at day 3 and around 40% was still positive by day 7. High estimates of submicroscopic residual parasitemia were also described in a recent study in Uganda, which found a day 7 prevalence of approximately 65% after AL or AL plus primaquine treatment, as detected by reverse transcription real-time PCR (qRT-PCR).11 In the present study, residual submicroscopic parasitemia was also shown by db-PCR-NALFIA, but the detected prevalence during follow-up was lower compared with qPCR. This is in line with expectations, because the LoD of db-PCR-NALFIA (∼1 parasite/μL) is higher than that of the qPCR used (∼0.2 parasites/μL).21

In agreement with other studies, the qPCR-based parasite density rapidly decreased for both AL and PA to < 2% of the enrollment value at day 1 and to around 0.02% of baseline at day 7.11,13 Remarkably, the PRR48 was found to be significantly higher for AL than for PA, indicating a faster clearance during AL treatment. This could be explained by the higher qPCR prevalence at day 2 in the PA group compared with the AL group. By contrast to the comparisons between relative densities, where only those positive at the follow-up day of interest were analyzed, all participants with day 0 and day 2 outcomes were included in the PRR48 analysis, with PRR48 estimates for those negative at day 2 arbitrarily set at a high value of 105. In the AL group, more participants were negative at day 2 and were thus assigned to this arbitrary value, resulting in a higher PRR48 for AL compared with PA-treated participants. A possible explanation for the difference in PRR48 between PA and AL is the once daily versus twice daily dosing, whereby the six-dose treatment regimen of AL allows for optimally effective drug concentrations over the complete treatment period, leading to faster clearance. However, this would be in contrast to microscopy-based studies that showed faster parasite clearance after PA compared with AL.32,33 A previous study found that having residual parasitemia at day 3 was associated with a lower PRR48.13 In the present study, this association could not be confirmed for participants with residual parasitemia at day 7, for both AL and PA.

Participants with qPCR-determined residual parasitemia at day 7 had a significantly higher gametocyte prevalence and density at enrollment and during follow-up, compared with those parasite-free at day 7. This confirms microscopy-based results from previous studies in which increased clearance times were found to be associated with a longer duration of gametocyte carriage.3436 Beshir et al.13 found that this association also exists for submicroscopic levels of residual parasitemia and gametocytes, which was confirmed in the present study. In addition, the study by Beshir et al.13 showed that the longer duration of gametocyte carriage in participants with residual parasitemia results in higher infectivity to mosquitos.

Residual parasitemia at day 7 after the start of treatment, as determined by db-PCR-NALFIA, was significantly associated with recurrent parasitemia up to 42 days. As expected, this association was stronger for recrudescent infections than for reinfections. This is in line with previous studies that found similar associations between microscopic positivity at day 2 and/or 3 and parasite recurrence.37,38 Other studies found this association at day 3 or 7 using molecular methods.13,18 However, both the present study and Chang et al.11 found no association between quantitative (reverse transcriptase) PCR [q(RT-)PCR] detected residual parasitemia and parasite recurrence. The difference between qPCR and db-PCR-NALFIA in this respect may be explained by the higher prevalence of residual parasitemia as detected by qPCR. It is unknown whether and to what extent the detected residual submicroscopic parasitemia reflects the presence of viable parasites, nonviable parasites or residual DNA circulating at very low densities.11 If a large proportion of low-density residual parasitemia consists of nonviable parasites or residual DNA, detected more frequently by qPCR than by db-PCR-NALFIA, the stronger the association between residual parasitemia and treatment outcome using db-PCR-NALFIA can be explained by differences in detection limits between the assays. This would imply that high analytical sensitivity of a diagnostic test does not necessarily translate into optimal clinical value of the method. It is also important to note that the association between residual parasitemia and treatment failure is likely to be stronger for recrudescences than for reinfections. The strength of this association, and thereby the applicability of molecular testing shortly after treatment to identify a potential treatment failure early, may therefore increase in areas with high levels of drug resistance.

To further establish the value of different molecular tools for the follow-up of patients after treatment, future studies should assess the viability of residual submicroscopic parasitemia after ACT treatment, comparing those with a recrudescence to those with reinfections and ACPR, for example by attempting to culture low-density residual parasites.11 Furthermore, the present study did not include membrane-feeding assays to assess whether the association between residual parasitemia and the presence of gametocyte mRNA indeed translates into an increased transmission potential, which would provide additional evidence that the longer duration of (submicroscopic) parasite carriage results in an increased infectivity to mosquitos. In this dataset, the recrudescence rate appeared to be fairly high, but the numbers are small and based on the detailed full trial analyses there are no indications that either of the ACTs was failing in this study. In addition, there is a well appreciated risk of overestimating the number of recrudescences in high transmission settings because of the high complexity of infections.39 Finally, the existence of associations between residual parasitemia and parasite recurrence, the preferred detection method and possible clinical implications need to be confirmed in future studies.

To conclude, this study demonstrates the detection of low-density parasitemia after microscopically successful ACT treatment, which confirms the findings from previous studies.1013,38,40 The data are relevant for the interpretation of clearance dynamics of both the relatively new PA and the commonly used AL. Both qPCR and db-PCR-NALFIA can be useful tools for patient follow-up after treatment, whereby in the clinical setting especially a negative db-PCR-NALFIA outcome at day 7 after treatment initiation may be a marker for an ACPR during further follow-up, keeping in mind that predictive values depend on the prevalence and may, thus, differ between settings.

Acknowledgments:

We thank the team of St. Jude’s Clinic, study participants, and their parents/guardians. We also thank Felix Choy for assisting with genotyping of samples from participants with recurrent parasitemia. Finally, we would like to thank Shin-Poong for providing pyronaridine–artesunate.

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Author Notes

Address correspondence to Johanna M. Roth, Department of Medical Microbiology, Academic Medical Center, Laboratory for Clinical Parasitology (L1-247), Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: j.m.roth@amc.uva.nl

Financial support: This work was supported by the EU FP7-Health-2013.0-1 project “Translation of the direct-on-blood PCR-NALFIA system into an innovative near point-of-care diagnostic for malaria” (DIAGMAL) (grant number 601714).

Disclosures: Shin Poong Pharmaceutical Company (Seoul, South-Korea) provided pyronaridine-artesunate tablets and granules, but had no further role in study design, data collection, data analysis and writing of the report.

Authors’ addresses: Johanna M. Roth, Menno D. de Jong, Henk D. F. H. Schallig, and Pètra F. Mens, Academic Medical Center, Amsterdam, The Netherlands, E-mails: j.m.roth@amc.uva.nl, m.d.dejong@amc.uva.nl, h.d.schallig@amc.uva.nl, and p.f.mens@amc.uva.nl. Patrick Sawa, George Omweri, and Nicodemus Makio, International Centre of Insect Physiology and Ecology, Mbita Point, Kenya, E-mails: psawa@icipe.org, gomweri@icipe.org, and nmakio@icipe.org. Victor Osoti, KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya, E-mail: vosoti@kemri-wellcome.org.

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