Investigation of an airport-associated cluster of falciparum malaria in Frankfurt, Germany, 2022.

Airport malaria is uncommon but increasing in Europe and often difficult to diagnose. We describe the clinical, epidemiological and environmental investigations of a cluster of airport malaria cases and measures taken in response. Three Frankfurt International Airport employees without travel histories to malaria-endemic areas were diagnosed with Plasmodium falciparum malaria in Germany in 2022. Two cases were diagnosed within 1 week, and the third one after 10 weeks. Two cases had severe disease, all three recovered fully. The cases worked in separate areas and no specific location for the transmissions could be identified. No additional cases were detected among airport employees. In June and July, direct flights from Equatorial Guinea, Nigeria and Angola and one parcel originating in Ghana arrived at Frankfurt airport. No vector-competent mosquitoes could be trapped to identify the source of the outbreak. Whole genome sequencing of P. falciparum genomes showed a high genetic relatedness between samples of the three cases and suggested the geographical origin closest to Ghana. A diagnosis of airport malaria should prompt appropriate and comprehensive outbreak investigations to identify the source and to prevent severe forms of falciparum malaria.

High Proportion of Genome-Wide Homology and Increased Pretreatment pvcrt Levels in Plasmodium vivax Late Recurrences: a Chloroquine Therapeutic Efficacy Study.

Chloroquine (CQ) is the first-line treatment for Plasmodium vivax malaria in most countries where malaria is endemic. Monitoring P. vivax CQ resistance (CQR) is critical but remains challenged by the difficulty to distinguish real treatment failure from reinfection or liver relapse. The therapeutic efficacy of CQ against uncomplicated P. vivax malaria was evaluated in Gia Lai Province, Vietnam. Sixty-seven patients were enrolled and followed for 42 days using microscopy and quantitative PCR. Adequate clinical and parasitological response (ACPR) was 100% (66/66) on day 28 but 75.4% (49/65) on day 42. Eighteen recurrences (27.7%) were detected, with a median time to recurrence of 42 days (interquartile range [IQR], 35 to 42) and blood CQ concentration of <100 ng/ml. Primary infections leading to recurrence occurred in younger individuals (median age for ACPR = 25 years [IQR, 20 to 28]; recurrences = 18 [16 to 21]; P = 0.002) had a longer parasite clearance time (PCT for ACPR = 47.5 h [IQR, 36.2 to 59.8 h]; recurrences = 54.2 [48.4 to 62.0]; P = 0.035) and higher pvcrt gene expression (median relative expression ratio for ACPR = 0.09 [IQR, 0.05 to 0.22]; recurrences = 0.20 [0.15 to 0.56]; P = 0.002), but showed no differences in ex vivo CQ sensitivity. Parasite genotyping by microsatellites, single nucleotide polymorphism (SNP) barcoding, and whole-genome sequencing (WGS) identified a majority of homologous recurrences, with 80% (8/10) showing >98% identity by descent to paired day 0 samples. This study shows that CQ remained largely efficacious to treat P. vivax in Gia Lai; i.e., recurrences occurred late (>day 28) and in the presence of low blood CQ concentrations. However, the combination of both WGS and gene expression analysis (pvcrt) data with clinical data (PCT) allowed us to identify potential emergence of low-grade CQR, which should be closely monitored. (This study has been registered at ClinicalTrials.gov under identifier NCT02610686.).

Efficacy of dihydroartemisinin/piperaquine and artesunate monotherapy for the treatment of uncomplicated Plasmodium falciparum malaria in Central Vietnam.

BACKGROUND: Artemisinin-based combination therapies (ACTs) have significantly contributed to reduce Plasmodium falciparum malaria burden in Vietnam, but their efficacy is challenged by treatment failure of dihydroartemisinin/piperaquine ACT in Southern provinces. OBJECTIVES: To assess the efficacy of dihydroartemisinin/piperaquine for uncomplicated P. falciparum malaria in Gia Lai, Central Vietnam, and determine parasite resistance to artemisinin (ClinicalTrials.gov identifier NCT02604966). METHODS: Sixty patients received either dihydroartemisinin/piperaquine (4 mg/kg/day, 3 days; n = 33) or artesunate monotherapy (4 mg/kg/day, 3 days; n = 27) followed by dihydroartemisinin/piperaquine (AS + DHA/PPQ). Clinical phenotypes were determined during a 42 day follow-up and analysed together with ex vivo susceptibility to antimalarials and molecular markers of drug resistance. RESULTS: Day 3 positivity rate was significantly higher in the AS + DHA/PPQ arm compared with dihydroartemisinin/piperaquine (70.4% versus 39.4%, P = 0.016). Parasite clearance time was 95.2 h (AS + DHA/PPQ) versus 71.9 h (dihydroartemisinin/piperaquine, P = 0.063) and parasite clearance half-life was 7.4 h (AS + DHA/PPQ) versus 7.0 h (dihydroartemisinin/piperaquine, P = 0.140). Adequate clinical and parasitological response at Day 42 was 100% in both arms. By RT-qPCR, 36% (19/53) patients remained positive until Day 7. No recurrences were detected. kelch13 artemisinin resistance mutations were found in 87% (39/45) of isolates and 50% (20/40) were KEL1/C580Y. The piperaquine resistance marker plasmepsin-2 was duplicated in 10.4% (5/48). Isolates from Day 3-positive patients (n = 18) had higher ex vivo survival rates to artemisinin compounds (P < 0.048) and prevalence of kelch13 mutations (P = 0.005) than Day 3-negative patients (n = 5). The WHO definition of artemisinin resistance was fulfilled in 60% (24/40) of cases. CONCLUSIONS: Although dihydroartemisinin/piperaquine remained effective to treat P. falciparum, the high Day 3 positivity rate and prevalence of KEL1 strains calls for continuous monitoring of dihydroartemisinin/piperaquine efficacy in Central Vietnam.

The epidemiology of Plasmodium falciparum and Plasmodium vivax in East Sepik Province, Papua New Guinea, pre- and post-implementation of national malaria control efforts.

BACKGROUND: In the past decade, national malaria control efforts in Papua New Guinea (PNG) have received renewed support, facilitating nationwide distribution of free long-lasting insecticidal nets (LLINs), as well as improvements in access to parasite-confirmed diagnosis and effective artemisinin-combination therapy in 2011-2012. METHODS: To study the effects of these intensified control efforts on the epidemiology and transmission of Plasmodium falciparum and Plasmodium vivax infections and investigate risk factors at the individual and household level, two cross-sectional surveys were conducted in the East Sepik Province of PNG; one in 2005, before the scale-up of national campaigns and one in late 2012-early 2013, after 2 rounds of LLIN distribution (2008 and 2011-2012). Differences between studies were investigated using Chi square (χ2), Fischer's exact tests and Student's t-test. Multivariable logistic regression models were built to investigate factors associated with infection at the individual and household level. RESULTS: The prevalence of P. falciparum and P. vivax in surveyed communities decreased from 55% (2005) to 9% (2013) and 36% to 6%, respectively. The mean multiplicity of infection (MOI) decreased from 1.8 to 1.6 for P. falciparum (p = 0.08) and from 2.2 to 1.4 for P. vivax (p < 0.001). Alongside these reductions, a shift towards a more uniform distribution of infections and illness across age groups was observed but there was greater heterogeneity across the study area and within the study villages. Microscopy positive infections and clinical cases in the household were associated with high rate infection households (> 50% of household members with Plasmodium infection). CONCLUSION: After the scale-up of malaria control interventions in PNG between 2008 and 2012, there was a substantial reduction in P. falciparum and P. vivax infection rates in the studies villages in East Sepik Province. Understanding the extent of local heterogeneity in malaria transmission and the driving factors is critical to identify and implement targeted control strategies to ensure the ongoing success of malaria control in PNG and inform the development of tools required to achieve elimination. In household-based interventions, diagnostics with a sensitivity similar to (expert) microscopy could be used to identify and target high rate households.

Efficacy of artemether-lumefantrine and dihydroartemisinin-piperaquine for the treatment of uncomplicated malaria in Papua New Guinea.

BACKGROUND: In 2009, the Papua New Guinea (PNG) Department of Health adopted artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DHA-PPQ) as the first- and second-line treatments for uncomplicated malaria, respectively. This study was conducted to assess the efficacy of both drugs following adoption of the new policy. METHODS: Between June 2012 and September 2014, a therapeutic efficacy study was conducted in East Sepik and Milne Bay Provinces of PNG in accordance with the standard World Health Organization (WHO) protocol for surveillance of anti-malarial drug efficacy. Patients ≥ 6 months of age with microscopy confirmed Plasmodium falciparum or Plasmodium vivax mono-infections were enrolled, treated with AL or DHA-PPQ, and followed up for 42 days. Study endpoints were adequate clinical and parasitological response (ACPR) on days 28 and 42. The in vitro efficacy of anti-malarials and the prevalence of selected molecular markers of resistance were also determined. RESULTS: A total of 274 P. falciparum and 70 P. vivax cases were enrolled. The day-42 PCR-corrected ACPR for P. falciparum was 98.1% (104/106) for AL and 100% (135/135) for DHA-PPQ. The day-42 PCR-corrected ACPR for P. vivax was 79.0% (15/19) for AL and 92.3% (36/39) for DHA-PPQ. Day 3 parasite clearance of P. falciparum was 99.2% with AL and 100% with DHA-PPQ. In vitro testing of 96 samples revealed low susceptibility to chloroquine (34% of samples above IC50 threshold) but not to lumefantrine (0%). Molecular markers assessed in a sub-set of the study population indicated high rates of chloroquine resistance in P. falciparum (pfcrt SVMNT: 94.2%, n = 104) and in P. vivax (pvmdr1 Y976F: 64.8%, n = 54). CONCLUSIONS: AL and DHA-PPQ were efficacious as first- and second-line treatments for uncomplicated malaria in PNG. Continued in vivo efficacy monitoring is warranted considering the threat of resistance to artemisinin and partner drugs in the region and scale-up of artemisinin-based combination therapy in PNG.

Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections.

BACKGROUND: Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. New amplicon sequencing and data analysis protocols are needed for genotyping in epidemiological studies and drug efficacy trials of P. falciparum. METHODS: Targeted sequencing of molecular marker csp and novel marker cpmp was conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol allowing to multiplex up to 384 samples in a single sequencing run was applied. Software "HaplotypR" was developed for data analysis. RESULTS: Cpmp was highly diverse (He = 0.96) in contrast to csp (He = 0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold. CONCLUSIONS: To reliably detect haplotypes at very low frequencies, experiments are best performed in duplicate and should aim for coverage of >10'000 reads/amplicon. When compared to length polymorphic marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority clones.

Accuracy of an HRP-2/panLDH rapid diagnostic test to detect peripheral and placental Plasmodium falciparum infection in Papua New Guinean women with anaemia or suspected malaria.

BACKGROUND: The diagnosis of malaria during pregnancy is complicated by placental sequestration, asymptomatic infection, and low-density peripheral parasitaemia. Where intermittent preventive treatment (IPT) with sulfadoxine-pyrimethamine is threatened by drug resistance, or is inappropriate due to low transmission, intermittent screening and treatment (ISTp) with rapid diagnostic tests for malaria (RDT) could be a valuable alternative. Therefore, the accuracy of RDTs to detect peripheral and placental infection was assessed in a declining transmission setting in Papua New Guinea (PNG). METHODS: The performance of a combination RDT detecting histidine-rich protein-2 (HRP-2) and Plasmodium lactate dehydrogenase (pLDH), and light microscopy (LM), to diagnose peripheral Plasmodium falciparum and Plasmodium vivax infections during pregnancy, were assessed using quantitative real-time PCR (qPCR) as the reference standard. Participants in a malaria prevention trial in PNG with a haemoglobin ≤90 g/L, or symptoms suggestive of malaria, were tested. Ability of RDT and LM to detect active placental infection on histology was evaluated in some participants. RESULTS: Among 876 women, 1162 RDTs were undertaken (anaemia: 854 [73.5 %], suspected malaria: 308 [26.5 %]). qPCR detected peripheral infection during 190 RDT episodes (165 P. falciparum, 19 P. vivax, 6 mixed infections). Overall, RDT detected peripheral P. falciparum infection with 45.6 % sensitivity (95 % CI 38.0-53.4), a specificity of 96.4 % (95.0-97.4), a positive predictive value of 68.4 % (59.1-76.8), and a negative predictive value of 91.1 % (89.2-92.8). RDT performance to detect P. falciparum was inferior to LM, more so amongst anaemic women (18.6 vs 45.3 % sensitivity, Liddell's exact test, P < 0.001) compared to symptomatic women (72.9 vs 82.4 % sensitivity, P = 0.077). RDT and LM missed 88.0 % (22/25) and 76.0 % (19/25) of P. vivax infections, respectively. In a subset of women tested at delivery and who had placental histology (n = 158) active placental infection was present in 19.6 %: all three peripheral blood infection detection methods (RDT, LM, qPCR) missed >50 % of these infections. CONCLUSIONS: In PNG, HRP-2/pLDH RDTs may be useful to diagnose peripheral P. falciparum infections in symptomatic pregnant women. However, they are not sufficiently sensitive for use in intermittent screening amongst asymptomatic (anaemic) women. These findings have implications for the management of malaria in pregnancy. The adverse impact of infections undetected by RDT or LM on pregnancy outcomes needs further evaluation.

Temporal changes in Plasmodium falciparum anti-malarial drug sensitivity in vitro and resistance-associated genetic mutations in isolates from Papua New Guinea.

BACKGROUND: In northern Papua New Guinea (PNG), most Plasmodium falciparum isolates proved resistant to chloroquine (CQ) in vitro between 2005 and 2007, and there was near-fixation of pfcrt K76T, pfdhfr C59R/S108N and pfmdr1 N86Y. To determine whether the subsequent introduction of artemisinin combination therapy (ACT) and reduced CQ-sulphadoxine-pyrimethamine pressure had attenuated parasite drug susceptibility and resistance-associated mutations, these parameters were re-assessed between 2011 and 2013. METHODS: A validated fluorescence-based assay was used to assess growth inhibition of 52 P. falciparum isolates from children in a clinical trial in Madang Province. Responses to CQ, lumefantrine, piperaquine, naphthoquine, pyronaridine, artesunate, dihydroartemisinin, artemether were assessed. Molecular resistance markers were detected using a multiplex PCR ligase detection reaction fluorescent microsphere assay. RESULTS: CQ resistance (in vitro concentration required for 50% parasite growth inhibition (IC50) >100 nM) was present in 19% of isolates. All piperaquine and naphthoquine IC50s were <100 nM and those for lumefantrine, pyronaridine and the artemisinin derivatives were in low nM ranges. Factor analysis of IC50s showed three groupings (lumefantrine; CQ, piperaquine, naphthoquine; pyronaridine, dihydroartemisinin, artemether, artesunate). Most isolates (96%) were monoclonal pfcrt K76T (SVMNT) mutants and most (86%) contained pfmdr1 N86Y (YYSND). No wild-type pfdhfr was found but most isolates contained wild-type (SAKAA) pfdhps. Compared with 2005-2007, the geometric mean (95% CI) CQ IC50 was lower (87 (71-107) vs 167 (141-197) nM) and there had been no change in the prevalence of pfcrt K76T or pfmdr1 mutations. There were fewer isolates of the pfdhps (SAKAA) wild-type (60 vs 100%) and pfdhfr mutations persisted. CONCLUSIONS: Reflecting less drug pressure, in vitro CQ sensitivity appears to be improving in Madang Province despite continued near-fixation of pfcrt K76T and pfmdr1 mutations. Temporal changes in IC50s for other anti-malarial drugs were inconsistent but susceptibility was preserved. Retention or increases in pfdhfr and pfdhps mutations reflect continued use of sulphadoxine-pyrimethamine in the study area including through paediatric intermittent preventive treatment. The susceptibility of local isolates to lumefantrine may be unrelated to those of other ACT partner drugs. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12610000913077 .

Artemisinin-naphthoquine versus artemether-lumefantrine for uncomplicated malaria in Papua New Guinean children: an open-label randomized trial.

BACKGROUND: Artemisinin combination therapies (ACTs) with broad efficacy are needed where multiple Plasmodium species are transmitted, especially in children, who bear the brunt of infection in endemic areas. In Papua New Guinea (PNG), artemether-lumefantrine is the first-line treatment for uncomplicated malaria, but it has limited efficacy against P. vivax. Artemisinin-naphthoquine should have greater activity in vivax malaria because the elimination of naphthoquine is slower than that of lumefantrine. In this study, the efficacy, tolerability, and safety of these ACTs were assessed in PNG children aged 0.5-5 y. METHODS AND FINDINGS: An open-label, randomized, parallel-group trial of artemether-lumefantrine (six doses over 3 d) and artemisinin-naphthoquine (three daily doses) was conducted between 28 March 2011 and 22 April 2013. Parasitologic outcomes were assessed without knowledge of treatment allocation. Primary endpoints were the 42-d P. falciparum PCR-corrected adequate clinical and parasitologic response (ACPR) and the P. vivax PCR-uncorrected 42-d ACPR. Non-inferiority and superiority designs were used for falciparum and vivax malaria, respectively. Because the artemisinin-naphthoquine regimen involved three doses rather than the manufacturer-specified single dose, the first 188 children underwent detailed safety monitoring. Of 2,542 febrile children screened, 267 were randomized, and 186 with falciparum and 47 with vivax malaria completed the 42-d follow-up. Both ACTs were safe and well tolerated. P. falciparum ACPRs were 97.8% and 100.0% in artemether-lumefantrine and artemisinin-naphthoquine-treated patients, respectively (difference 2.2% [95% CI -3.0% to 8.4%] versus -5.0% non-inferiority margin, p = 0.24), and P. vivax ACPRs were 30.0% and 100.0%, respectively (difference 70.0% [95% CI 40.9%-87.2%], p<0.001). Limitations included the exclusion of 11% of randomized patients with sub-threshold parasitemias on confirmatory microscopy and direct observation of only morning artemether-lumefantrine dosing. CONCLUSIONS: Artemisinin-naphthoquine is non-inferior to artemether-lumefantrine in PNG children with falciparum malaria but has greater efficacy against vivax malaria, findings with implications in similar geo-epidemiologic settings within and beyond Oceania. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12610000913077. Please see later in the article for the Editors' Summary.

Genomic surveillance of malaria parasites in an indigenous community in the Peruvian Amazon.

Hard-to-reach communities represent Peru's main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = 0.07-0.52 and Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ's Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.

Genomic surveillance of malaria parasites in an indigenous community in the Peruvian Amazon.

Hard-to-reach communities represent Peru's main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = -0.52 & Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ's Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.

Antigen persistence of rapid diagnostic tests in pregnant women in Nanoro, Burkina Faso, and the implications for the diagnosis of malaria in pregnancy.

OBJECTIVES: To evaluate persistence of several Plasmodium antigens in pregnant women after treatment and compare diagnostics during treatment follow-up. METHODS: Thirty-two pregnant women (N = 32) with confirmed malaria infection by a histidine-rich protein 2 (HRP2)-based rapid diagnostic test (RDT) and microscopy were followed for 28 days after artemisinin-based combination therapy (ACT). A Plasmodium lactate dehydrogenase (pLDH)-based RDT and two ELISAs based on the detection of dihydrofolate reductase-thymidylate synthase (DHFR-TS) and haeme detoxification protein (HDP) were compared with each other and to RT-PCR at each visit. RESULTS: The mean visit number (95% confidence interval) on which the HRP2-based RDT was still positive after treatment was 3.4 (2.7-4.1) visits with some patients still positive at day 28. This is significantly later than the pLDH-based RDT [0.84 (0.55-1.1)], microscopy (median 1, range 1-3), DHFR-TS-ELISA [1.7 (1.1-2.3)] and RT-PCR (median 2, range 1-5) (P < 0.05), but not significantly later than HDP-ELISA [2.1 (1.6-2.7)]. Lower gravidity and higher parasite density at day 0 resulted in significantly longer positive results with most tests (P < 0.05). CONCLUSIONS: HRP2 can persist up to 28 days after ACT treatment; therefore, this test is not suitable for treatment follow-up in pregnant women and can generate problems when using this test during intermittent preventive treatment (IPTp). DHFR-TS is less persistent than HRP2, making it a potentially interesting target for diagnosis.

Relative contribution of low-density and asymptomatic infections to Plasmodium vivax transmission in the Amazon: pooled analysis of individual participant data from population-based cross-sectional surveys.

Background: Low-density and asymptomatic Plasmodium vivax infections remain largely undetected and untreated and may contribute significantly to malaria transmission in the Amazon. Methods: We analysed individual participant data from population-based surveys that measured P vivax prevalence by microscopy and polymerase chain reaction (PCR) between 2002 and 2015 and modelled the relationship between parasite density and infectiousness to vectors using membrane feeding assay data. We estimated the proportion of sub-patent (i.e., missed by microscopy) and asymptomatic P vivax infections and examined how parasite density relates to clinical manifestations and mosquito infection in Amazonian settings. Findings: We pooled 24,986 observations from six sites in Brazil and Peru. P vivax was detected in 6·8% and 2·1% of them by PCR and microscopy, respectively. 58·5% to 92·6% of P vivax infections were asymptomatic and 61·2% to 96·3% were sub-patent across study sites. P vivax density thresholds associated with clinical symptoms were one order of magnitude higher in children than in adults. We estimate that sub-patent parasite carriers are minimally infectious and contribute 12·7% to 24·9% of the community-wide P vivax transmission, while asymptomatic carriers are the source of 28·2% to 79·2% of mosquito infections. Interpretation: Asymptomatic P vivax carriers constitute a vast infectious reservoir that, if targeted by malaria elimination strategies, could substantially reduce malaria transmission in the Amazon. Infected children may remain asymptomatic despite high parasite densities that elicit clinical manifestations in adults. Funding: US National Institutes of Health, Fundação de Amparo à Pesquisa do Estado de São Paulo, and Belgium Development Cooperation.

Systematic review and meta-analysis: rapid diagnostic tests versus placental histology, microscopy and PCR for malaria in pregnant women.

BACKGROUND: During pregnancy, malaria infection with Plasmodium falciparum or Plasmodium vivax is related to adverse maternal health and poor birth outcomes. Diagnosis of malaria, during pregnancy, is complicated by the absence or low parasite densities in peripheral blood. Diagnostic methods, other than microscopy, are needed for detection of placental malaria. Therefore, the diagnostic accuracy of rapid diagnostic tests (RDTs), detecting antigen, and molecular techniques (PCR), detecting DNA, for the diagnosis of Plasmodium infections in pregnancy was systematically reviewed. METHODS: MEDLINE, EMBASE and Web of Science were searched for studies assessing the diagnostic accuracy of RDTs, PCR, microscopy of peripheral and placental blood and placental histology for the detection of malaria infection (all species) in pregnant women. RESULTS: The results of 49 studies were analysed in metandi (Stata), of which the majority described P. falciparum infections. Although both placental and peripheral blood microscopy cannot reliably replace histology as a reference standard for placental P. falciparum infection, many studies compared RDTs and PCR to these tests. The proportion of microscopy positives in placental blood (sensitivity) detected by peripheral blood microscopy, RDTs and PCR are respectively 72% [95% CI 62-80], 81% [95% CI 55-93] and 94% [95% CI 86-98]. The proportion of placental blood microscopy negative women that were negative in peripheral blood microscopy, RDTs and PCR (specificity) are 98% [95% CI 95-99], 94% [95% CI 76-99] and 77% [95% CI 71-82]. Based on the current data, it was not possible to determine if the false positives in RDTs and PCR are caused by sequestered parasites in the placenta that are not detected by placental microscopy. CONCLUSION: The findings suggest that RDTs and PCR may have good performance characteristics to serve as alternatives for the diagnosis of malaria in pregnancy, besides any other limitations and practical considerations concerning the use of these tests. Nevertheless, more studies with placental histology as reference test are urgently required to reliably determine the accuracy of RDTs and PCR for the diagnosis of placental malaria. P. vivax-infections have been neglected in diagnostic test accuracy studies of malaria in pregnancy.

Longitudinal tracking and quantification of individual Plasmodium falciparum clones in complex infections.

Longitudinal tracking of individual Plasmodium falciparum strains in multi-clonal infections is essential for investigating infection dynamics of malaria. The traditional genotyping techniques did not permit tracking changes in individual clone density during persistent natural infections. Amplicon deep sequencing (Amp-Seq) offers a tool to address this knowledge gap. The sensitivity of Amp-Seq for relative quantification of clones was investigated using three molecular markers, ama1-D2, ama1-D3, and cpmp. Amp-Seq and length-polymorphism based genotyping were compared for their performance in following minority clones in longitudinal samples from Papua New Guinea. Amp-Seq markers were superior to length-polymorphic marker msp2 in detecting minority clones (sensitivity Amp-Seq: 95%, msp2: 85%). Multiplicity of infection (MOI) by Amp-Seq was 2.32 versus 1.73 for msp2. The higher sensitivity had no effect on estimates of force of infection because missed minority clones were detected in preceding or succeeding bleeds. Individual clone densities were tracked longitudinally by Amp-Seq despite MOI > 1, thus providing an additional parameter for investigating malaria infection dynamics. Amp-Seq based genotyping of longitudinal samples improves detection of minority clones and estimates of MOI. Amp-Seq permits tracking of clone density over time to study clone competition or the dynamics of specific, i.e. resistance-associated genotypes.

New developments in malaria diagnostics: monoclonal antibodies against Plasmodium dihydrofolate reductase-thymidylate synthase, heme detoxification protein and glutamate rich protein.

Currently available rapid diagnostic tests (RDTs) for malaria show large variation in sensitivity and specificity, and there are concerns about their stability under field conditions. To improve current RDTs, monoclonal antibodies (mAbs) for novel malaria antigens have been developed and screened for their possible use in new diagnostic tests. Three antigens, glutamate rich protein (GLURP), dihydrofolate reductase-thymidylate synthase (DHFR-TS) and heme detoxification protein (HDP), were selected based on literature searches. Recombinant antigens were produced and used to immunize mice. Antibody-producing cell lines were subsequently selected and the resulting antibodies were screened for specificity against Plasmodium falciparum and Plasmodium vivax. The most optimal antibody couples were selected based on antibody affinity (expressed as dissociation constants, KD) and detection limit of crude antigen extract from P. falciparum 3D7 culture. The highest affinity antibodies have KD values of 0.10 nM ± 0.014 (D5) and 0.068 ± 0.015 nM (D6) for DHFR-TS mAbs, 0.10 ± 0.022 nM (H16) and 0.21 ± 0.022 nM (H18) for HDP mAbs and 0.11 ± 0.028 nM (G23) and 0.33 ± 0.093 nM (G22) for GLURP mAbs. The newly developed antibodies performed at least as well as commercially available histidine rich protein antibodies (KD of 0.16 ± 0.13 nM for PTL3 and 1.0 ± 0.049 nM for C1-13), making them promising reagents for further test development.

Evaluation of antigen detection tests, microscopy, and polymerase chain reaction for diagnosis of malaria in peripheral blood in asymptomatic pregnant women in Nanoro, Burkina Faso.

Rapid diagnostics tests (RDTs) detect malaria specific antigen(s) in the circulation, even when parasites are sequestered in the placenta and not visible by microscopy. However, research on their diagnostic accuracy during pregnancy is limited. Pregnant women (n = 418) were screened for malaria during routine antenatal care by using two RDTs that detect histidine-rich protein 2 (HRP2) or Plasmodium lactate dehydrogenase, and enzyme-linked immunosorbent assays with antibodies that detect dihydrofolate reductase-thymidylate synthase or heme-detoxification protein, and compared with real-time polymerase chain reaction (RT-PCR) and microscopy for evaluation of their diagnostic accuracy. Prevalence of malaria infection was high (53% by PCR). The RT-PCR and the HRP2 RDT detected most cases of malaria during pregnancy, whereas microscopy, the Plasmodium lactate dehydrogenase RDT, and enzyme-linked immunosorbent assays for dihydrofolate reductase-thymidylate synthase and heme-detoxification protein antibodies did not detect several low-density infections. Therefore, the HRP2 RDT could be a useful tool in high-transmission areas for diagnosis of malaria in asymptomatic pregnant women.