Risk of Malaria Following Untreated Subpatent Plasmodium falciparum Infections: Results Over 4 Years From a Cohort in a High-Transmission Area in Western Kenya.

BACKGROUND: People with suspected malaria may harbor Plasmodium falciparum undetected by rapid diagnostic test (RDT). The impact of these subpatent infections on the risk of developing clinical malaria is not fully understood. METHODS: We analyzed subpatent P. falciparum infections using a longitudinal cohort in a high-transmission site in Kenya. Weighted Kaplan-Meier models estimated the risk difference (RD) for clinical malaria during the 60 days following a symptomatic subpatent infection. Stratum-specific estimates by age and transmission season assessed modification. RESULTS: Over 54 months, we observed 1128 symptomatic RDT-negative suspected malaria episodes, of which 400 (35.5%) harbored subpatent P. falciparum. Overall, the 60-day risk of developing clinical malaria was low following all episodes (8.6% [95% confidence interval, 6.7%-10.4%]). In the low-transmission season, the risk of clinical malaria was slightly higher in those with subpatent infection, whereas the opposite was true in the high-transmission season (low-transmission season RD, 2.3% [95% confidence interval, .4%-4.2%]; high-transmission season RD, -4.8% [-9.5% to -.05%]). CONCLUSIONS: The risk of developing clinical malaria among people with undetected subpatent infections is low. A slightly elevated risk in the low-transmission season may merit alternate management, but RDTs identify clinically relevant infections in the high-transmission season.

Micro-heterogeneity of transmission shapes submicroscopic malaria carriage in coastal Tanzania.

BACKGROUND: Asymptomatic carriage of malaria parasites persists even as malaria transmission declines. Low density infections are often submicroscopic, not detected by rapid diagnostic tests (RDTs) or microscopy, but detectable by PCR. METHODS: To characterize submicroscopic Plasmodium falciparum carriage in an area of declining malaria transmission, asymptomatic persons >5 years of age in rural Bagamoyo District, Tanzania, were screened using RDT, microscopy, and PCR. We investigated the size of the submicroscopic reservoir of infection across villages, determined factors associated with submicroscopic carriage, and assessed the natural history of submicroscopic malaria over four weeks. RESULTS: Among 6,076 participants, P. falciparum prevalence by RDT, microscopy, and PCR was 9%, 9%, and 28%, respectively, with roughly two-thirds of PCR-positive individuals harboring submicroscopic infection. Adult status, female gender, dry season months, screened windows, and bednet use were associated with submicroscopic carriage. Among 15 villages encompassing 80% of participants, the proportion of submicroscopic carriers increased with decreasing village-level malaria prevalence. Over four weeks, 23% (61/266) of submicroscopic carriers became RDT-positive, with half exhibiting symptoms, while half (133/266) were no longer parasitemic at the end of four weeks. Progression to RDT-positive patent malaria occurred more frequently in villages with higher malaria prevalence. CONCLUSIONS: Micro-heterogeneity in transmission observed at the village level appears to impact both the size of the submicroscopic reservoir and the likelihood of submicroscopic carriers developing patent malaria in coastal Tanzania.

Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.

BACKGROUND: While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. METHODS: Multiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo. RESULTS: Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min. CONCLUSION: Combined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.

Similar Prevalence of Plasmodium falciparum and Non-P. falciparum Malaria Infections among Schoolchildren, Tanzania1.

Achieving malaria elimination requires considering both Plasmodium falciparum and non-P. falciparum infections. We determined prevalence and geographic distribution of 4 Plasmodium spp. by performing PCR on dried blood spots collected within 8 regions of Tanzania during 2017. Among 3,456 schoolchildren, 22% had P. falciparum, 24% had P. ovale spp., 4% had P. malariae, and 0.3% had P. vivax infections. Most (91%) schoolchildren with P. ovale infections had low parasite densities; 64% of P. ovale infections were single-species infections, and 35% of those were detected in low malaria endemic regions. P. malariae infections were predominantly (73%) co-infections with P. falciparum. P. vivax was detected mostly in northern and eastern regions. Co-infections with >1 non-P. falciparum species occurred in 43% of P. falciparum infections. A high prevalence of P. ovale infections exists among schoolchildren in Tanzania, underscoring the need for detection and treatment strategies that target non-P. falciparum species.

Household Transmission of Influenza A Viruses in 2021-2022.

Importance: Influenza virus infections declined globally during the COVID-19 pandemic. Loss of natural immunity from lower rates of influenza infection and documented antigenic changes in circulating viruses may have resulted in increased susceptibility to influenza virus infection during the 2021-2022 influenza season. Objective: To compare the risk of influenza virus infection among household contacts of patients with influenza during the 2021-2022 influenza season with risk of influenza virus infection among household contacts during influenza seasons before the COVID-19 pandemic in the US. Design, Setting, and Participants: This prospective study of influenza transmission enrolled households in 2 states before the COVID-19 pandemic (2017-2020) and in 4 US states during the 2021-2022 influenza season. Primary cases were individuals with the earliest laboratory-confirmed influenza A(H3N2) virus infection in a household. Household contacts were people living with the primary cases who self-collected nasal swabs daily for influenza molecular testing and completed symptom diaries daily for 5 to 10 days after enrollment. Exposures: Household contacts living with a primary case. Main Outcomes and Measures: Relative risk of laboratory-confirmed influenza A(H3N2) virus infection in household contacts during the 2021-2022 season compared with prepandemic seasons. Risk estimates were adjusted for age, vaccination status, frequency of interaction with the primary case, and household density. Subgroup analyses by age, vaccination status, and frequency of interaction with the primary case were also conducted. Results: During the prepandemic seasons, 152 primary cases (median age, 13 years; 3.9% Black; 52.0% female) and 353 household contacts (median age, 33 years; 2.8% Black; 54.1% female) were included and during the 2021-2022 influenza season, 84 primary cases (median age, 10 years; 13.1% Black; 52.4% female) and 186 household contacts (median age, 28.5 years; 14.0% Black; 63.4% female) were included in the analysis. During the prepandemic influenza seasons, 20.1% (71/353) of household contacts were infected with influenza A(H3N2) viruses compared with 50.0% (93/186) of household contacts in 2021-2022. The adjusted relative risk of A(H3N2) virus infection in 2021-2022 was 2.31 (95% CI, 1.86-2.86) compared with prepandemic seasons. Conclusions and Relevance: Among cohorts in 5 US states, there was a significantly increased risk of household transmission of influenza A(H3N2) in 2021-2022 compared with prepandemic seasons. Additional research is needed to understand reasons for this association.

Household Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 in the United States: Living Density, Viral Load, and Disproportionate Impact on Communities of Color.

BACKGROUND: Households are hot spots for severe acute respiratory syndrome coronavirus 2 transmission. METHODS: This prospective study enrolled 100 coronavirus disease 2019 (COVID-19) cases and 208 of their household members in North Carolina though October 2020, including 44% who identified as Hispanic or non-White. Households were enrolled a median of 6 days from symptom onset in the index case. Incident secondary cases within the household were detected using quantitative polymerase chain reaction of weekly nasal swabs (days 7, 14, 21) or by seroconversion at day 28. RESULTS: Excluding 73 household contacts who were PCR-positive at baseline, the secondary attack rate (SAR) among household contacts was 32% (33 of 103; 95% confidence interval [CI], 22%-44%). The majority of cases occurred by day 7, with later cases confirmed as household-acquired by viral sequencing. Infected persons in the same household had similar nasopharyngeal viral loads (intraclass correlation coefficient = 0.45; 95% CI, .23-.62). Households with secondary transmission had index cases with a median viral load that was 1.4 log10 higher than those without transmission (P = .03), as well as higher living density (more than 3 persons occupying fewer than 6 rooms; odds ratio, 3.3; 95% CI, 1.02-10.9). Minority households were more likely to experience high living density and had a higher risk of incident infection than did White households (SAR, 51% vs 19%; P = .01). CONCLUSIONS: Household crowding in the context of high-inoculum infections may amplify the spread of COVID-19, potentially contributing to disproportionate impact on communities of color.

Classification of disease recurrence using transition likelihoods with expectation-maximization algorithm.

When an infectious disease recurs, it may be due to treatment failure or a new infection. Being able to distinguish and classify these two different outcomes is critical in effective disease control. A multi-state model based on Markov processes is a typical approach to estimating the transition probability between the disease states. However, it can perform poorly when the disease state is unknown. This article aims to demonstrate that the transition likelihoods of baseline covariates can distinguish one cause from another with high accuracy in infectious diseases such as malaria. A more general model for disease progression can be constructed to allow for additional disease outcomes. We start from a multinomial logit model to estimate the disease transition probabilities and then utilize the baseline covariate's transition information to provide a more accurate classification result. We apply the expectation-maximization (EM) algorithm to estimate unknown parameters, including the marginal probabilities of disease outcomes. A simulation study comparing our classifier to the existing two-stage method shows that our classifier has better accuracy, especially when the sample size is small. The proposed method is applied to determining relapse vs reinfection outcomes in two Plasmodium vivax treatment studies from Cambodia that used different genotyping approaches to demonstrate its practical use.

Distribution and Temporal Dynamics of Plasmodium falciparum Chloroquine Resistance Transporter Mutations Associated With Piperaquine Resistance in Northern Cambodia.

BACKGROUND: Newly emerged mutations within the Plasmodium falciparum chloroquine resistance transporter (PfCRT) can confer piperaquine resistance in the absence of amplified plasmepsin II (pfpm2). In this study, we estimated the prevalence of co-circulating piperaquine resistance mutations in P. falciparum isolates collected in northern Cambodia from 2009 to 2017. METHODS: The sequence of pfcrt was determined for 410 P. falciparum isolates using PacBio amplicon sequencing or whole genome sequencing. Quantitative polymerase chain reaction was used to estimate pfpm2 and pfmdr1 copy number. RESULTS: Newly emerged PfCRT mutations increased in prevalence after the change to dihydroartemisinin-piperaquine in 2010, with >98% of parasites harboring these mutations by 2017. After 2014, the prevalence of PfCRT F145I declined, being outcompeted by parasites with less resistant, but more fit PfCRT alleles. After the change to artesunate-mefloquine, the prevalence of parasites with amplified pfpm2 decreased, with nearly half of piperaquine-resistant PfCRT mutants having single-copy pfpm2. CONCLUSIONS: The large proportion of PfCRT mutants that lack pfpm2 amplification emphasizes the importance of including PfCRT mutations as part of molecular surveillance for piperaquine resistance in this region. Likewise, it is critical to monitor for amplified pfmdr1 in these PfCRT mutants, as increased mefloquine pressure could lead to mutants resistant to both drugs.

Selection of Cytochrome b Mutants Is Rare among Plasmodium falciparum Patients Failing Treatment with Atovaquone-Proguanil in Cambodia.

Atovaquone-proguanil remains effective against multidrug-resistant Plasmodium falciparum in Southeast Asia, but resistance is mediated by a single point mutation in cytochrome b (cytb) that can arise during treatment. Among 14 atovaquone-proguanil treatment failures in a clinical trial in Cambodia, only one recrudescence harbored the cytb mutation Y268C. Deep sequencing did not detect the mutation at baseline or in the first 3 days of treatment, suggesting that it arose de novo Further sequencing across cytb similarly found no low-frequency cytb mutations that were up-selected from baseline to recrudescence. Copy number amplification in dihydroorotate dehydrogenase (DHODH) and cytb as markers of atovaquone tolerance was also absent. Cytb mutation played a minor role in atovaquone-proguanil treatment failures in an active comparator clinical trial.

Efficient Transmission of Mixed Plasmodium falciparum/vivax Infections From Humans to Mosquitoes.

BACKGROUND: In Southeast Asia, people are often coinfected with different species of malaria (Plasmodium falciparum [Pf] and Plasmodium vivax [Pv]) as well as with multiple clones of the same species. Whether particular species or clones within mixed infections are more readily transmitted to mosquitoes remains unknown. METHODS: Laboratory-reared Anopheles dirus were fed on blood from 119 Pf-infected Cambodian adults, with 5950 dissected to evaluate for transmitted infection. Among 12 persons who infected mosquitoes, polymerase chain reaction and amplicon deep sequencing were used to track species and clone-specific transmission to mosquitoes. RESULTS: Seven of 12 persons that infected mosquitoes harbored mixed Pf/Pv infection. Among these 7 persons, all transmitted Pv with 2 transmitting both Pf and Pv, leading to Pf/Pv coinfection in 21% of infected mosquitoes. Up to 4 clones of each species were detected within persons. Shifts in clone frequency were detected during transmission. However, in general, all parasite clones in humans were transmitted to mosquitoes, with individual mosquitoes frequently carrying multiple transmitted clones. CONCLUSIONS: Malaria diversity in human hosts was maintained in the parasite populations recovered from mosquitoes fed on their blood. However, in persons with mixed Pf/Pv malaria, Pv appears to be transmitted more readily, in association with more prevalent patent gametocytemia.

Relapse or reinfection: Classification of malaria infection using transition likelihoods.

In patients with Plasmodium vivax malaria treated with effective blood-stage therapy, the recurrent illness may occur due to relapse from latent liver-stage infection or reinfection from a new mosquito bite. Classification of the recurrent infection as either relapse or reinfection is critical when evaluating the efficacy of an anti-relapse treatment. Although one can use whether a shared genetic variant exists between baseline and recurrence genotypes to classify the outcome, little has been suggested to use both sharing and nonsharing variants to improve the classification accuracy. In this paper, we develop a novel classification criterion that utilizes transition likelihoods to distinguish relapse from reinfection. When tested in extensive simulation experiments with known outcomes, our classifier has superior operating characteristics. A real data set from 78 Cambodian P. vivax malaria patients was analyzed to demonstrate the practical use of our proposed method.

Selective sweep suggests transcriptional regulation may underlie Plasmodium vivax resilience to malaria control measures in Cambodia.

Cambodia, in which both Plasmodium vivax and Plasmodium falciparum are endemic, has been the focus of numerous malaria-control interventions, resulting in a marked decline in overall malaria incidence. Despite this decline, the number of P vivax cases has actually increased. To understand better the factors underlying this resilience, we compared the genetic responses of the two species to recent selective pressures. We sequenced and studied the genomes of 70 P vivax and 80 P falciparum isolates collected between 2009 and 2013. We found that although P falciparum has undergone population fracturing, the coendemic P vivax population has grown undisrupted, resulting in a larger effective population size, no discernable population structure, and frequent multiclonal infections. Signatures of selection suggest recent, species-specific evolutionary differences. Particularly, in contrast to P falciparum, P vivax transcription factors, chromatin modifiers, and histone deacetylases have undergone strong directional selection, including a particularly strong selective sweep at an AP2 transcription factor. Together, our findings point to different population-level adaptive mechanisms used by P vivax and P falciparum parasites. Although population substructuring in P falciparum has resulted in clonal outgrowths of resistant parasites, P vivax may use a nuanced transcriptional regulatory approach to population maintenance, enabling it to preserve a larger, more diverse population better suited to facing selective threats. We conclude that transcriptional control may underlie P vivax's resilience to malaria control measures. Novel strategies to target such processes are likely required to eradicate P vivax and achieve malaria elimination.

Association of a Novel Mutation in the Plasmodium falciparum Chloroquine Resistance Transporter With Decreased Piperaquine Sensitivity.

Background: Amplified copy number in the plasmepsin II/III genes within Plasmodium falciparum has been associated with decreased sensitivity to piperaquine. To examine this association and test whether additional loci might also contribute, we performed a genome-wide association study of ex vivo P. falciparum susceptibility to piperaquine. Methods: Plasmodium falciparum DNA from 183 samples collected primarily from Cambodia was genotyped at 33716 genome-wide single nucleotide polymorphisms (SNPs). Linear mixed models and random forests were used to estimate associations between parasite genotypes and piperaquine susceptibility. Candidate polymorphisms were evaluated for their association with dihydroartemisinin-piperaquine treatment outcomes in an independent dataset. Results: Single nucleotide polymorphisms on multiple chromosomes were associated with piperaquine 90% inhibitory concentrations (IC90) in a genome-wide analysis. Fine-mapping of genomic regions implicated in genome-wide analyses identified multiple SNPs in linkage disequilibrium with each other that were significantly associated with piperaquine IC90, including a novel mutation within the gene encoding the P. falciparum chloroquine resistance transporter, PfCRT. This mutation (F145I) was associated with dihydroartemisinin-piperaquine treatment failure after adjusting for the presence of amplified plasmepsin II/III, which was also associated with decreased piperaquine sensitivity. Conclusions: Our data suggest that, in addition to plasmepsin II/III copy number, other loci, including pfcrt, may also be involved in piperaquine resistance.

Microscopic Plasmodium falciparum Gametocytemia and Infectivity to Mosquitoes in Cambodia.

Although gametocytes are essential for malaria transmission, in Africa many falciparum-infected persons without smear-detectable gametocytes still infect mosquitoes. To see whether the same is true in Southeast Asia, we determined the infectiousness of 119 falciparum-infected Cambodian adults to Anopheles dirus mosquitoes by membrane feeding. Just 5.9% of subjects infected mosquitoes. The 8.4% of patients with smear-detectable gametocytes were >20 times more likely to infect mosquitoes than those without and were the source of 96% of all mosquito infections. In low-transmission settings, targeting transmission-blocking interventions to those with microscopic gametocytemia may have an outsized effect on malaria control and elimination.

Using Amplicon Deep Sequencing to Detect Genetic Signatures of Plasmodium vivax Relapse.

Plasmodium vivax infections often recur due to relapse of hypnozoites from the liver. In malaria-endemic areas, tools to distinguish relapse from reinfection are needed. We applied amplicon deep sequencing to P. vivax isolates from 78 Cambodian volunteers, nearly one-third of whom suffered recurrence at a median of 68 days. Deep sequencing at a highly variable region of the P. vivax merozoite surface protein 1 gene revealed impressive diversity-generating 67 unique haplotypes and detecting on average 3.6 cocirculating parasite clones within individuals, compared to 2.1 clones detected by a combination of 3 microsatellite markers. This diversity enabled a scheme to classify over half of recurrences as probable relapses based on the low probability of reinfection by multiple recurring variants. In areas of high P. vivax diversity, targeted deep sequencing can help detect genetic signatures of relapse, key to evaluating antivivax interventions and achieving a better understanding of relapse-reinfection epidemiology.

Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance.

Cambodia's first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC50] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure.

Attenuation of Plasmodium falciparum in vitro drug resistance phenotype following culture adaptation compared to fresh clinical isolates in Cambodia.

BACKGROUND: There is currently no standardized approach for assessing in vitro anti-malarial drug susceptibility. Potential alterations in drug susceptibility results between fresh immediate ex vivo (IEV) and cryopreserved culture-adapted (CCA) Plasmodium falciparum isolates, as well as changes in parasite genotype during culture adaptation were investigated. METHODS: The 50 % inhibitory concentration (IC50) of 12 P. falciparum isolates from Cambodia against a panel of commonly used drugs were compared using both IEV and CCA. Results were compared using both histidine-rich protein-2 ELISA (HRP-2) and SYBR-Green I fluorescence methods. Molecular genotyping and amplicon deep sequencing were also used to compare multiplicity of infection and genetic polymophisms in fresh versus culture-adapted isolates. RESULTS: IC50 for culture-adapted specimens were significantly lower compared to the original fresh isolates for both HRP-2 and SYBR-Green I assays, with greater than a 50 % decline for the majority of drug-assay combinations. There were correlations between IC50s from IEV and CCA for most drugs assays. Infections were nearly all monoclonal, with little or no change in merozoite surface protein 1 (MSP1), MSP2, glutamate-rich protein (GLURP) or apical membrane antigen 1 (AMA1) polymorphisms, nor differences in P. falciparum multidrug resistance 1 gene (PfMDR1) copy number or single nucleotide polymorphisms following culture adaptation. CONCLUSIONS: The overall IC50 reduction combined with the correlation between fresh isolates and culture-adapted drug susceptibility assays suggests the utility of both approaches, as long as there is consistency of method, and remaining mindful of possible attenuation of resistance phenotype occurring in culture. Further study should be done in higher transmission settings where polyclonal infections are prevalent.

Field evaluation of a real-time fluorescence loop-mediated isothermal amplification assay, RealAmp, for the diagnosis of malaria in Thailand and India.

BACKGROUND: To eliminate malaria, surveillance for submicroscopic infections is needed. Molecular methods can detect submicroscopic infections but have not hitherto been amenable to implementation in surveillance programs. A portable loop-mediated isothermal amplification assay called RealAmp was assessed in 2 areas of low malaria transmission. METHODS: RealAmp was evaluated in 141 patients from health clinics in India (passive surveillance) and in 127 asymptomatic persons in Thailand (active surveillance). The diagnostic validity, precision, and predictive value of RealAmp were determined using polymerase chain reaction (PCR) as the reference method. A pilot study of RealAmp was also performed on samples from patients presenting at a Thai health center. RESULTS: A total of 96 and 7 positive cases were detected in India and Thailand, respectively, via PCR. In comparison with nested PCR, the sensitivity and specificity of RealAmp in India were 94.8% (95% confidence interval [CI], 88.3%-98.3%) and 100% (95% CI, 92.1%-100%), respectively, with correct identification of all 5 Plasmodium vivax cases. In Thailand, compared with pooled real-time PCR, RealAmp demonstrated 100% sensitivity (95% CI, 59.0%-100%) and 96.7% specificity (95% CI, 91.7%-99.1%). Testing at the health center demonstrated RealAmp's potential to serve as a point-of-care test with results available in 30-75 minutes. CONCLUSION: RealAmp was comparable to PCR in detecting malaria parasites and shows promise as a tool to detect submicroscopic infections in malaria control and elimination programs worldwide.