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

Abstract

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 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 ( = 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 ( = 0.692). qPCR-determined parasitemia at day 7 was associated with increased prevalence and density of gametocytes at baseline ( = 0.014 and = 0.003, for prevalence and density, respectively) and during follow-up ( = 0.007 and = 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, = 0.003), but this association was not found for qPCR (OR: 0.701, 95% CI: 0.312-1.578, = 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.

Parasite clearance, cure rate, post-treatment prophylaxis and safety of standard 3-day versus an extended 6-day treatment of artemether–lumefantrine and a single low-dose primaquine for uncomplicated Plasmodium falciparum malaria in Bagamoyo district, Tanzania: a randomized controlled trial

“…The highly sensitive PCR methods used in this study may partly explain the increase in D3 PCR positivity over time, as compared with previous studies from the same site [16]. Other studies in Uganda and Kenya also report high PCR positivity after ACT [16,54,55]. The study in Uganda linked D3 P. falciparum PCR positivity with the presence of asexual ring stages and mature gametocytes, but without increased risk to treatment failure [56].…”

Detection of Plasmodium falciparum by Light Microscopy, Loop-Mediated Isothermal Amplification, and Polymerase Chain Reaction on Day 3 after Initiation of Artemether–Lumefantrine Treatment for Uncomplicated Malaria in Bagamoyo District, Tanzania: A Comparative Trial

“…19 This may contribute to the lower specificity of LAMP compared with PCR, although DNA contamination could also be a potential issue with sensitive molecular methods. 17 Other studies conducted in Kenya 6 and Uganda 20 reported up to 62% and 76% P. falciparum positivity by qPCR on day 3 after ACT. In the Ugandan study, day 3 P. falciparum PCR positivity was associated with detection of persisting asexual ring stage parasites and mature gametocytes using specific molecular markers, but without increased risk to treatment failure within the 28-day follow-up.…”

“…5 We believe these findings require careful consideration, especially because other reports on persistent submicroscopic parasitemia after ACT have shown association with treatment failure, longer gametocyte carriage, and subsequently higher transmission potential. 6,7 The WHO recommends therapeutic efficacy studies (TES) to be conducted with regular intervals as part of programmatic drug resistance surveillance in malaria-endemic countries. 3 In TES, finger-prick blood samples are collected and examined by light microscopy on a daily basis for determining parasite clearance up to day 3 after initiation of ACT treatment.…”

Six decades of lateral flow immunoassay: from determining metabolic markers to diagnosing COVID-19

“…Over the past 10–15 years, a significant number of platforms have been proposed that have been developed for the detection of HA fragments using lateral flow technology and PCR [121] – [125] . These methods have simplified PCR technology, eliminating electrophoresis for confirming the presence of nucleic acid after DNA amplification, and of purchase of expensive equipment for molecular genetic analysis [126] – [128] .…”