Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and ...rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1-8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed.
There is a critical need for developing new malaria diagnostic tools that are sensitive, cost effective and capable of performing large scale diagnosis. The real-time PCR methods are particularly ...robust for large scale screening and they can be used in malaria control and elimination programs. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. falciparum and the Plasmodium genus by real-time PCR. A total of 119 samples consisting of different malaria species and mixed infections were used to test the utility of the novel PET-PCR primers in the diagnosis of clinical samples. The sensitivity and specificity were calculated using a nested PCR as the gold standard and the novel primer sets demonstrated 100% sensitivity and specificity. The limits of detection for P. falciparum was shown to be 3.2 parasites/µl using both Plasmodium genus and P. falciparum-specific primers and 5.8 parasites/µl for P. ovale, 3.5 parasites/µl for P. malariae and 5 parasites/µl for P. vivax using the genus specific primer set. Moreover, the reaction can be duplexed to detect both Plasmodium spp. and P. falciparum in a single reaction. The PET-PCR assay does not require internal probes or intercalating dyes which makes it convenient to use and less expensive than other real-time PCR diagnostic formats. Further validation of this technique in the field will help to assess its utility for large scale screening in malaria control and elimination programs.
Antimalarial drug resistance has historically arisen through convergent
mutations in
parasite populations in Southeast Asia and South America. For the past decade in Southeast Asia, artemisinins, the ...core component of first-line antimalarial therapies, have experienced delayed parasite clearance associated with several
mutations, primarily C580Y. We report that mutant
has emerged independently in Guyana, with genome analysis indicating an evolutionary origin distinct from Southeast Asia.
C580Y parasites were observed in 1.6% (14/854) of samples collected in Guyana in 2016-2017. Introducing
C580Y or R539T mutations by gene editing into local parasites conferred high levels of
artemisinin resistance.
growth competition assays revealed a fitness cost associated with these
variants, potentially explaining why these resistance alleles have not increased in frequency more quickly in South America. These data place local malaria control efforts at risk in the Guiana Shield.
Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference ...laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method.
Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples.
This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.
Accurate diagnosis of malaria infections continues to be challenging and elusive, especially in the detection of submicroscopic infections. Developing new malaria diagnostic tools that are sensitive ...enough to detect low-level infections, user friendly, cost effective and capable of performing large scale diagnosis, remains critical. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. ovale by real-time PCR. In our study, a total of 173 clinical samples, consisting of different malaria species, were utilized to test this novel PET-PCR primer. The sensitivity and specificity were calculated using nested-PCR as the reference test. The novel primer set demonstrated a sensitivity of 97.5% and a specificity of 99.2% (95% CI 85.2-99.8% and 95.2-99.9% respectively). Furthermore, the limit of detection for P. ovale was found to be 1 parasite/μl. The PET-PCR assay is a new molecular diagnostic tool with comparable performance to other commonly used PCR methods. It is relatively easy to perform, and amiable to large scale malaria surveillance studies and malaria control and elimination programs. Further field validation of this novel primer will be helpful to ascertain the utility for large scale malaria screening programs.
Partial artemisinin resistance is suspected if delayed parasite clearance (ie, persistence of parasitaemia on day 3 after treatment initiation) is observed. Validated markers of artemisinin partial ...resistance in southeast Asia, Plasmodium falciparum kelch13 (Pfkelch13) R561H and P574L, have been reported in Rwanda but no association with parasite clearance has been observed. We aimed to establish the efficacy of artemether–lumefantrine and genetic characterisation of Pfkelch13 alleles and their association with treatment outcomes.
This open-label, single-arm, multicentre, therapeutic efficacy study was done in 2018 in three Rwandan sites: Masaka, Rukara, and Bugarama. Children aged 6–59 months with P falciparum monoinfection and fever were eligible and treated with a 3-day course of artemether–lumefantrine. Treatment response was monitored for 28 days using weekly microscopy screenings of blood samples for P falciparum. Mutations in Pfkelch13 and P falciparum multidrug resistance-1 (Pfmdr1) genes were characterised in parasites collected from enrolled participants. Analysis of flanking microsatellites surrounding Pfkelch13 was done to define the origins of the R561H mutations. The primary endpoint was PCR-corrected parasitological cure on day 28, as per WHO protocol.
228 participants were enrolled and 224 (98·2%) reached the study endpoint. PCR-corrected efficacies were 97·0% (95% CI 88–100) in Masaka, 93·8% (85–98) in Rukara, and 97·2% (91–100) in Bugarama. Pfkelch13 R561H mutations were present in 28 (13%) of 218 pre-treatment samples and P574L mutations were present in two (1%) pre-treatment samples. 217 (90%) of the 240 Pfmdr1 haplotypes observed in the pretreatment samples, had either the NFD (N86Y, Y184F, D1246Y) or NYD haplotype. Eight (16%) of 51 participants in Masaka and 12 (15%) of 82 participants in Rukara were microscopically positive 3 days after treatment initiation, which was associated with pre-treatment presence of Pfkelch13 R561H in Masaka (p=0·0005). Genetic analysis of Pfkelch13 R561H mutations suggest their common ancestry and local origin in Rwanda.
We confirm evidence of emerging artemisinin partial resistance in Rwanda. Although artemether–lumefantrine remains efficacious, vigilance for decreasing efficacy, further characterisation of artemisinin partial resistance, and evaluation of additional antimalarials in Rwanda should be considered.
The US President's Malaria Initiative.
For the French translation of the abstract see Supplementary Materials section.
Recent efforts in malaria control have resulted in great gains in reducing the burden of Plasmodium falciparum, but P. vivax has been more refractory. Its ability to form dormant liver stages ...confounds control and elimination efforts. To compare the efficacy and safety of primaquine regimens for radical cure, we undertook a randomized controlled trial in Ethiopia.
Patients with normal glucose-6-phosphate dehydrogenase status with symptomatic P. vivax mono-infection were enrolled and randomly assigned to receive either chloroquine (CQ) or artemether-lumefantrine (AL), alone or in combination with 14 d of semi-supervised primaquine (PQ) (3.5 mg/kg total). A total of 398 patients (n = 104 in the CQ arm, n = 100 in the AL arm, n = 102 in the CQ+PQ arm, and n = 92 in the AL+PQ arm) were followed for 1 y, and recurrent episodes were treated with the same treatment allocated at enrolment. The primary endpoints were the risk of P. vivax recurrence at day 28 and at day 42. The risk of recurrent P. vivax infection at day 28 was 4.0% (95% CI 1.5%-10.4%) after CQ treatment and 0% (95% CI 0%-4.0%) after CQ+PQ. The corresponding risks were 12.0% (95% CI 6.8%-20.6%) following AL alone and 2.3% (95% CI 0.6%-9.0%) following AL+PQ. On day 42, the risk was 18.7% (95% CI 12.2%-28.0%) after CQ, 1.2% (95% CI 0.2%-8.0%) after CQ+PQ, 29.9% (95% CI 21.6%-40.5%) after AL, and 5.9% (95% CI 2.4%-13.5%) after AL+PQ (overall p < 0.001). In those not prescribed PQ, the risk of recurrence by day 42 appeared greater following AL treatment than CQ treatment (HR = 1.8 95% CI 1.0-3.2; p = 0.059). At the end of follow-up, the incidence rate of P. vivax was 2.2 episodes/person-year for patients treated with CQ compared to 0.4 for patients treated with CQ+PQ (rate ratio: 5.1 95% CI 2.9-9.1; p < 0.001) and 2.3 episodes/person-year for AL compared to 0.5 for AL+PQ (rate ratio: 6.4 95% CI 3.6-11.3; p < 0.001). There was no difference in the occurrence of adverse events between treatment arms. The main limitations of the study were the early termination of the trial and the omission of haemoglobin measurement after day 42, resulting in an inability to estimate the cumulative risk of anaemia.
Despite evidence of CQ-resistant P. vivax, the risk of recurrence in this study was greater following treatment with AL unless it was combined with a supervised course of PQ. PQ combined with either CQ or AL was well tolerated and reduced recurrence of vivax malaria by 5-fold at 1 y.
ClinicalTrials.gov NCT01680406.
Plasmodium vivax infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional ...molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries. Our laboratory has recently developed a new platform called RealAmp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of malaria parasites. Here we describe new primers for the detection of P. vivax using the RealAmp method. Three pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the P. vivax genome. The amplification was carried out at 64°C using SYBR Green or SYTO-9 intercalating dyes for 90 minutes with the tube scanner set to collect fluorescence signals at 1-minute intervals. Clinical samples of P. vivax and other human-infecting malaria parasite species were used to determine the sensitivity and specificity of the primers by comparing with an 18S ribosomal RNA-based nested PCR as the gold standard. The new set of primers consistently detected laboratory-maintained isolates of P. vivax from different parts of the world. The primers detected P. vivax in the clinical samples with 94.59% sensitivity (95% CI: 87.48-98.26%) and 100% specificity (95% CI: 90.40-100%) compared to the gold standard nested-PCR method. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting P. vivax.
In this study, the performance of a commercially available malaria LAMP assay (Alethia® Malaria Plus LAMP) was evaluated using retrospective clinical samples obtained from travelers returning to the ...United States of America (USA). Recently, several laboratories in non-malaria endemic countries evaluated the use of the loop mediated isothermal amplification (LAMP) assays for the diagnosis of imported malaria cases. These tests are simpler than polymerase-chain reaction (PCR)-based assays and were shown to have high sensitivity. Much of malaria diagnoses in the USA, is undertaken at the state level using mainly microscopy and rapid diagnostic tests (RDTs). However, molecular tools offer greater sensitivity over microscopy and RDTs. A reliable, easy to perform molecular assay can provide a test of choice for the accurate detection of malaria parasites in places where expert microscopy is lacking and/or for the detection of low-parasite density infections.
The Alethia® Malaria Plus LAMP assay was easy to use, had similar test performances as the real-time PCR reference test and results were obtained faster (within 1 h) than the reference test. The sensitivity of the assay was 100% with a kappa score of 1 when compared to the reference PET-PCR assay.
The state of Roraima, in Brazil, has recently seen an increase in the number of reported Plasmodium falciparum infections believed to be imported from neighboring countries. The objective of this ...study was to determine the prevalence of Plasmodium species among patients attending malaria health posts in Roraima and quantify the infections attributable to imported malaria. This cross-sectional case study was carried out between March 2016 and September 2018. Study participants were recruited as they exited the malaria health post. Information about residence, occupation and travel history was collected using a questionnaire. A dried blood spot was collected and used for malaria diagnosis by PCR. A total of 1222 patients were enrolled. Of the 80% Plasmodium positive samples, 50% were P. falciparum, 34% P. vivax, 8% mixed P. falciparum/P. vivax and 0.2% mixed P. falciparum/P. ovale infections and 8% tested positive for Plasmodium, but the species could not be identified. 80% of the malaria patients likely acquired infections in Venezuela and the remaining 20% acquired in Guyana, Brazil, Suriname and French Guyana. 50% of the study participants reported to be working in a mine. Results from this study support the hypothesis that imported malaria contribute to the bulk of malaria diagnosed in Roraima. These findings are in keeping with previous findings and should be considered when developing malaria control interventions.
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