Evaluating Plasmodium falciparum automatic detection and parasitemia estimation: A comparative study on thin blood smear images.

Malaria is a deadly disease that is transmitted through mosquito bites. Microscopists use a microscope to examine thin blood smears at high magnification (1000x) to identify parasites in red blood cells (RBCs). Estimating parasitemia is essential in determining the severity of the Plasmodium falciparum infection and guiding treatment. However, this process is time-consuming, labor-intensive, and subject to variation, which can directly affect patient outcomes. In this retrospective study, we compared three methods for measuring parasitemia from a collection of anonymized thin blood smears of patients with Plasmodium falciparum obtained from the Clinical Department of Parasitology-Mycology, National Reference Center (NRC) for Malaria in Paris, France. We first analyzed the impact of the number of field images on parasitemia count using our framework, MALARIS, which features a top-classifier convolutional neural network (CNN). Additionally, we studied the variation between different microscopists using two manual techniques to demonstrate the need for a reliable and reproducible automated system. Finally, we included thin blood smear images from an additional 102 patients to compare the performance and correlation of our system with manual microscopy and flow cytometry. Our results showed strong correlations between the three methods, with a coefficient of determination between 0.87 and 0.92.

Screening of malaria infections in human blood samples with varying parasite densities and anaemic conditions using AI-Powered mid-infrared spectroscopy.

BACKGROUND: Effective testing for malaria, including the detection of infections at very low densities, is vital for the successful elimination of the disease. Unfortunately, existing methods are either inexpensive but poorly sensitive or sensitive but costly. Recent studies have shown that mid-infrared spectroscopy coupled with machine learning (MIRs-ML) has potential for rapidly detecting malaria infections but requires further evaluation on diverse samples representative of natural infections in endemic areas. The aim of this study was, therefore, to demonstrate a simple AI-powered, reagent-free, and user-friendly approach that uses mid-infrared spectra from dried blood spots to accurately detect malaria infections across varying parasite densities and anaemic conditions. METHODS: Plasmodium falciparum strains NF54 and FCR3 were cultured and mixed with blood from 70 malaria-free individuals to create various malaria parasitaemia and anaemic conditions. Blood dilutions produced three haematocrit ratios (50%, 25%, 12.5%) and five parasitaemia levels (6%, 0.1%, 0.002%, 0.00003%, 0%). Dried blood spots were prepared on Whatman™ filter papers and scanned using attenuated total reflection-Fourier Transform Infrared (ATR-FTIR) for machine-learning analysis. Three classifiers were trained on an 80%/20% split of 4655 spectra: (I) high contrast (6% parasitaemia vs. negative), (II) low contrast (0.00003% vs. negative) and (III) all concentrations (all positive levels vs. negative). The classifiers were validated with unseen datasets to detect malaria at various parasitaemia levels and anaemic conditions. Additionally, these classifiers were tested on samples from a population survey in malaria-endemic villages of southeastern Tanzania. RESULTS: The AI classifiers attained over 90% accuracy in detecting malaria infections as low as one parasite per microlitre of blood, a sensitivity unattainable by conventional RDTs and microscopy. These laboratory-developed classifiers seamlessly transitioned to field applicability, achieving over 80% accuracy in predicting natural P. falciparum infections in blood samples collected during the field survey. Crucially, the performance remained unaffected by various levels of anaemia, a common complication in malaria patients. CONCLUSION: These findings suggest that the AI-driven mid-infrared spectroscopy approach holds promise as a simplified, sensitive and cost-effective method for malaria screening, consistently performing well despite variations in parasite densities and anaemic conditions. The technique simply involves scanning dried blood spots with a desktop mid-infrared scanner and analysing the spectra using pre-trained AI classifiers, making it readily adaptable to field conditions in low-resource settings. In this study, the approach was successfully adapted to field use, effectively predicting natural malaria infections in blood samples from a population-level survey in Tanzania. With additional field trials and validation, this technique could significantly enhance malaria surveillance and contribute to accelerating malaria elimination efforts.

Late Parasitological Failure and Subsequent Isolated Gametocytemia of Uncomplicated Plasmodium falciparum Malaria in a Returned Traveler From Ghana, 2023.

Herein, we report a case of uncomplicated falciparum malaria with late parasitological failure in a 45-year-old businessman returning from Ghana. The patient visited the emergency department with high fever, headache, and dizziness. He traveled without antimalarial chemoprophylaxis. Laboratory tests led to the diagnosis of uncomplicated falciparum malaria with an initial density of 37,669 parasites per µL of blood (p/µL). The patient was treated with intravenous artesunate followed by atovaquone/proguanil. He was discharged with improved condition and decreased parasite density of 887 p/µL. However, at follow-up, parasite density increased to 7,630 p/µL despite the absence of any symptoms. Suspecting treatment failure, the patient was administered intravenous artesunate and doxycycline for seven days and then artemether/lumefantrine for three days. Blood smear was negative for asexual parasitemia after re-treatment but positive for gametocytemia until day 101 from the initial diagnosis. Overall, this case highlights the risk of late parasitological failure in patients with imported uncomplicated falciparum malaria.

Estimation of parasitaemia in imported falciparum malaria using the results of a combined rapid diagnostic test. No big help from haematological parameters.

BACKGROUND: Microscopy continues to be the mainstay for the evaluation of parasitaemia in malaria but requires laboratory support and microbiological experience. Other fast and simple methods are necessary. METHODS: A retrospective observational study of imported malaria treated from July-2007 to December-2020 was carried out to evaluate the association between the degree of parasitaemia and both rapid diagnostic tests (RDT) reactivity patterns and haematological parameters. Plasmodium falciparum monoinfections diagnosed by peripheral blood smear and/or polymerase chain reaction (PCR),which also had a positive RDT result in the same blood sample, were included in the study. RESULTS: A total of 273 patients were included. Most of them were male (n = 256; 93.8%) and visiting friends and relatives (VFR) travellers (n = 252; 92.3%). Patients with plasmodial lactate dehydrogenase (pLDH) or aldolase and histidine-rich protein 2 (HRP-2) co-reactivity (Pan/Pf pattern) had a parasitaemia range between 0 and 37% while those with just HRP-2 reactivity (P. falciparum pattern) had ranges between 0 and 1%. Not a single case of P. falciparum pattern was found for parasitaemia ranges greater than 1%, showing a negative predictive value of 100% for high parasitaemia. All the correlations between haematological parameters and parasitaemia resulted to be weak, with a maximum rho coefficient of -0.35 for lymphocytes and platelets, and of 0.40 for neutrophils-to-lymphocytes count ratio. Multivariate predictive models were constructed reflecting a poor predictive capacity. CONCLUSIONS: The reactivity pattern of RDT allows a rapid semi-quantitative assessment of P. falciparum parasitaemia in travellers with imported malaria, discriminating patients with lower parasite loads. Haematological parameters were not able to estimate parasitaemia with sufficient precision.

Artemisinin-based combination therapy successfully treated two hyperparasitaemic Plasmodium falciparum cases.

Hyperparasitaemia is an important event in the cascade of Plasmodium falciparum severe malaria (SM), and may also lead to SM associated complications and death, if left untreated. Here, we report two hyperparasitaemic patients with no life-threatening complications. Malaria diagnosis was performed using thick and thin blood smears and immunochromatographic-based rapid diagnostic tests (RDTs) purchased from three different manufacturers. Parasitaemia was calculated following the World Health Organization (WHO) guidelines. Haematological and biochemical investigations were also performed. Weekly follow-up of blood smear examination, blood pressure and temperature were recorded up to day 63. The first patient had 42% parasitaemia (100% asexual parasites). The second patient had 9.5% parasitaemia, comprising 46% asexual and 54% sexual stages, with a 1:1 male to female ratio. On the day of admission, both had presented abnormal haematological and biochemical parameters compared to the reference values. Remarkably, both the patients recovered successfully with oral artemisinin-based combination therapy (ACT) and a single dose of primaquine on day 1. Weekly follow-up did not show any parasite suggesting successful treatment with ACT without any side effects. The presence of hypergametocytaemia may hinder malaria elimination efforts, if not treated immediately.

Performance of a novel melting curve-based qPCR assay for malaria parasites in routine clinical practice in non-endemic setting.

BACKGROUND: High-quality malaria diagnosis is essential for effective treatment and clinical disease management. Microscopy and rapid diagnostic tests are the conventional methods performed as first-line malaria diagnostics in non-endemic countries. However, these methods lack the characteristic to detect very low parasitaemia, and accurate identification of the Plasmodium species can be difficult. This study evaluated the performance of the MC004 melting curve-based qPCR for the diagnosis of malaria in routine clinical practice in non-endemic setting. METHODS AND RESULTS: Whole blood samples were collected from 304 patients with clinical suspicion of malaria and analysed by both the MC004 assay and conventional diagnostics. Two discrepancies were found between the MC004 assay and microscopy. Repeated microscopic analysis confirmed the qPCR results. Comparison of the parasitaemia of nineteen Plasmodium falciparum samples determined by both microscopy and qPCR showed the potential of the MC004 assay to estimate the parasite load of P. falciparum. Eight Plasmodium infected patients were followed after anti-malarial treatment by the MC004 assay and microscopy. The MC004 assay still detected Plasmodium DNA although no parasites were seen with microscopy in post-treatment samples. The rapid decline in Plasmodium DNA showed the potential for therapy-monitoring. CONCLUSION: Implementation of the MC004 assay in non-endemic clinical setting improved the diagnosis of malaria. The MC004 assay demonstrated superior Plasmodium species identification, the ability to indicate the Plasmodium parasite load, and can potentially detect submicroscopic Plasmodium infections.

CMOS Spectrophotometric Microsystem for Malaria Detection.

OBJECTIVES: Optical spectrophotometry has been explored to quantify Plasmodium falciparum malaria parasites at low parasitemia, with potential to overcome the limitations of detection in the current diagnostic methods. This work presents the design, simulation and fabrication of a CMOS microelectronic detection system to automatically quantify the presence of malaria parasites in a blood sample. METHODS: The designed system is composed by an array of 16 n+/p-substrate silicon junction photodiodes as photodetectors and 16 current to frequency (IF) converters. An optical setup was used to individually and jointly characterize the entire system. RESULTS: The IF converter was simulated and characterized in Cadence Tools using UMC 1180 MM/RF technology rules, featuring a resolution of 0.01 nA, a linearity up to 1800 nA and a sensitivity of 4430 Hz/nA. After fabrication in a silicon foundry, the photodiodes' characterization presented a responsivity peak of 120 mA/W (λ = 570 nm) and a dark current of 7.15 pA at 0 V. Regarding the IF converter, it exhibited high linearity (R2 ≈ 0.999) up to 30 nA, with a sensitivity of 4840 Hz/nA. Furthermore, the microsystem performance was validated using RBCs (Red Blood Cells) infected with P. falciparum and diluted at different parasitemia (12, 25 and 50 parasites/µL). CONCLUSION: The microsystem was able to distinguish between healthy and infected RBCs, with a sensitivity of 4.5 Hz/parasites.µL-1. SIGNIFICANCE: The developed microsystem presents a competitive result, when compared to the gold standard diagnosis methods, with increased potential for malaria in field diagnosis.

Measurement of Babesia bovis infected red blood cells using flow cytometry.

Rapid and accurate tools are needed for high-throughput in vitro antibabesial drug testing. In this study, flow cytometry for the measuring of Babesia bovis in vitro culture, was developed using SYBR Green I and compared against the results of fluorescence-based assay and microscopic assay. A high correlation of measured parasitemia was observed with high R2 value (R2 = 0.9991) between flow cytometry and microscopic analysis. The degree of antibabesial drug sensitivity against B. bovis determined by flow cytometry was 0.424 ± 0.173 µM. Similar to the results of previously published studies involving fluorescence spectrometry-based assay (0.408 ± 0.011 µM) and microscopy-based assay (0.400 ± 0.017 µM). The outcomes of this present study suggest that flow cytometry assay using SYBR Green I can potentially be useful in determining parasitemia and can serve as a rapid alternative method to antibabesial drug testing.

Longitudinal and Cross-sectional Analyses of Asymptomatic HIV-1/Malaria Co-infection in Kisumu County, Kenya.

Individuals infected with HIV-1 experience more frequent and more severe episodes of malaria and are likely to harbor asymptomatic parasitemia, thus potentially making them more efficient reservoirs of malaria. Two studies (cross-sectional and longitudinal) were designed in sequence between 2015-2018 and 2018-2020, respectively, to test the hypothesis that HIV-1 infected individuals have higher prevalence of asymptomatic parasitemia and gametocytemia than the HIV-1 negatives. This article describes the overall design of the two studies, encompassing data for the longitudinal study and additional data to the previously published baseline data for the cross-sectional study. In the cross-sectional study, HIV-1 positive participants were significantly older, more likely to be male, and more likely to have parasitemia relative to HIV-1 negatives (P < 0.01). In the longitudinal study, 300 participants were followed for 6 months. Of these, 102 were HIV-1 negative, 106 were newly diagnosed HIV-1 positive, and 92 were HIV-1 positive and on antiretroviral therapy, including antifolates, at enrollment. Overall parasitemia positivity at enrollment was 17.3% (52/300). Of these, 44% (23/52) were HIV-1 negative, 52% (27/52) were newly diagnosed HIV-1 positives, and only 4% (2/52) were HIV-1 positive and on treatment. Parasitemia for those on stable antiretroviral therapy was significantly lower (hazard ratio: 0.51, P < 0.001), compared with the HIV-1-negatives. On follow-up, there was a significant decline in parasitemia prevalence (hazard ratio: 0.74, P < 0.001) among the HIV patients newly initiated on antiretroviral therapy including trimethoprim-sulfamethoxasole. These data highlight the impact of HIV-1 and HIV treatment on asymptomatic parasitemia over time.

Paper functionalization for detection of Plasmodium falciparum DNA using square waves voltammetry.

Malaria elimination is a major goal to be reached in the next decade. Significant progress were made in the past, and the prevalence decreased in many areas while the positive trend stalled in the last years. The exact number of asymptomatic carriers of Plasmodium parasites is unknown since this population is not detected by conventional diagnosis methods and participate in the maintenance of transmission. Molecular methods to detect low parasitemia are not available at point-of-care in low-income countries of malaria endemic areas. Adaptation of molecular methods such as loop-mediated isothermal amplification of DNA may provide effective tools but it required simplification of DNA detection. Square waves voltammetry, easily imbedded in small device such as cell phone, was largely described for DNA detection but support for reaction was an issue to address. Here we used an efficient functionalization method of paper-based material to facilitate the interactions between isothermal amplification product and methylene blue for easy-to-use DNA detection. The proof-of-concept of qualitative detection of very low parasitemia from malaria infected patients using newly chemically treated paper for square waves voltammetry was obtained with a sensitivity and specificity of 100% and a limit-of-detection of 0.1 parasite. µL-1 corresponding to a parasitemia of 0.000002%.

Novel application of one-step pooled molecular testing and maximum likelihood approaches to estimate the prevalence of malaria parasitaemia among rapid diagnostic test negative samples in western Kenya.

BACKGROUND: Detection of malaria parasitaemia in samples that are negative by rapid diagnostic tests (RDTs) requires resource-intensive molecular tools. While pooled testing using a two-step strategy provides a cost-saving alternative to the gold standard of individual sample testing, statistical adjustments are needed to improve accuracy of prevalence estimates for a single step pooled testing strategy. METHODS: A random sample of 4670 malaria RDT negative dried blood spot samples were selected from a mass testing and treatment trial in Asembo, Gem, and Karemo, western Kenya. Samples were tested for malaria individually and in pools of five, 934 pools, by one-step quantitative polymerase chain reaction (qPCR). Maximum likelihood approaches were used to estimate subpatent parasitaemia (RDT-negative, qPCR-positive) prevalence by pooling, assuming poolwise sensitivity and specificity was either 100% (strategy A) or imperfect (strategy B). To improve and illustrate the practicality of this estimation approach, a validation study was constructed from pools allocated at random into main (734 pools) and validation (200 pools) subsets. Prevalence was estimated using strategies A and B and an inverse-variance weighted estimator and estimates were weighted to account for differential sampling rates by area. RESULTS: The prevalence of subpatent parasitaemia was 14.5% (95% CI 13.6-15.3%) by individual qPCR, 9.5% (95% CI (8.5-10.5%) by strategy A, and 13.9% (95% CI 12.6-15.2%) by strategy B. In the validation study, the prevalence by individual qPCR was 13.5% (95% CI 12.4-14.7%) in the main subset, 8.9% (95% CI 7.9-9.9%) by strategy A, 11.4% (95% CI 9.9-12.9%) by strategy B, and 12.8% (95% CI 11.2-14.3%) using inverse-variance weighted estimator from poolwise validation. Pooling, including a 20% validation subset, reduced costs by 52% compared to individual testing. CONCLUSIONS: Compared to individual testing, a one-step pooled testing strategy with an internal validation subset can provide accurate prevalence estimates of PCR-positivity among RDT-negatives at a lower cost.

Metagenomic Sequencing for the Diagnosis of Plasmodium spp. with Different Levels of Parasitemia in EDTA Blood of Malaria Patients-A Proof-of-Principle Assessment.

Molecular diagnostic approaches are increasingly included in the diagnostic workup and even in the primary diagnosis of malaria in non-endemic settings, where it is difficult to maintain skillful microscopic malaria detection due to the rarity of the disease. Pathogen-specific nucleic acid amplification, however, bears the risk of overlooking other pathogens associated with febrile illness in returnees from the tropics. Here, we assessed the discriminatory potential of metagenomic sequencing for the identification of different Plasmodium species with various parasitemia in EDTA blood of malaria patients. Overall, the proportion of Plasmodium spp.-specific sequence reads in the assessed samples showed a robust positive correlation with parasitemia (Spearman r = 0.7307, p = 0.0001) and a robust negative correlation with cycle threshold (Ct) values of genus-specific real-time PCR (Spearman r = -0.8626, p ≤ 0.0001). Depending on the applied bioinformatic algorithm, discrimination on species level was successful in 50% (11/22) to 63.6% (14/22) instances. Limiting factors for the discrimination on species level were very low parasitemia, species-depending lacking availability of reliable reference genomes, and mixed infections with high variance of the proportion of the infecting species. In summary, metagenomic sequencing as performed in this study is suitable for the detection of malaria in human blood samples, but the diagnostic detection limit for a reliable discrimination on species level remains higher than for competing diagnostic approaches like microscopy and PCR.

Automated measurement of malaria parasitaemia among asymptomatic blood donors in Malawi using the Sysmex XN-31 analyser: could such data be used to complement national malaria surveillance in real time?

BACKGROUND: The recent worldwide increase in malaria cases highlights the need for renewed efforts to eliminate malaria. The World Health Organization advocates that malaria surveillance becomes a core intervention. Current methods to estimate the malaria burden rely on clinical malaria case reports and surveys of asymptomatic parasite infection mainly from children < 5 years. In this study the hypothesis was that screening blood donors for malaria parasites would provide real-time information on the asymptomatic reservoir of parasites in the adult population and mirror other surveillance data. METHODS: This study was conducted in Malawi, a high malaria burden country, at the Malawi Blood Transfusion Service, which collects blood units at donation sites countrywide. A secondary analysis was conducted on data obtained from a prior Sysmex XN-31 analyser malaria diagnostic evaluation study utilizing residual donor blood samples. XN-31 malaria results, donor age, sex, geographical location, and collection date, were analysed using standard statistical methods. RESULTS: The malaria parasite prevalence in blood donors was 11.6% (614/5281 samples) increasing seasonally from December (8.6%) to April (18.3%). The median age was 21 years and 45.9% of donors were from urban areas, which showed a lower prevalence compared to non-urban regions. The Central administrative region had the highest and the Northern region the lowest malaria parasite prevalence. The donors were predominantly male (80.2%), 13.1% of whom had malaria parasites, which was significantly higher (p < 0.0001) than for female donors (7.4%). Multivariable logistic regression analysis showed that age, location, and collection month were significant predictors of malaria positivity in males, whereas in females only location was significant. There was no gender difference in parasite density nor gametocyte carriage. CONCLUSIONS: This study demonstrates the powerful utility of screening blood donors for malaria parasites using the XN-31, which not only improves the safety of blood transfusion, but provides valuable complementary surveillance data for malaria control, especially targeting males, who are generally excluded from periodic household surveys. Blood donations are sourced countrywide, year-round, and thus provide dynamic, real-time information on the malaria burden. Furthermore, the XN-31 identifies the asymptomatic human reservoir of infectious gametocytes, which must be targeted to eliminate malaria.

Parasitemia Levels in Trypanosoma cruzi Infection in Spain, an Area Where the Disease Is Not Endemic: Trends by Different Molecular Approaches.

Trypanosoma cruzi infection has expanded globally through human migration. In Spain, the mother-to-child route is the mode of transmission contributing to autochthonous Chagas disease (CD); however, most people acquired the infection in their country of origin and were diagnosed in the chronic phase (imported chronic CD). In this context, we assessed the quantitative potential of the Loopamp Trypanosoma cruzi detection kit (Sat-TcLAMP) based on satellite DNA (Sat-DNA) to determine parasitemia levels compared to those detected by real-time quantitative PCRs (qPCRs) targeting Sat-DNA (Sat-qPCR) and kinetoplast DNA minicircles (kDNA-qPCR). This study included 173 specimens from 39 autochthonous congenital and 116 imported chronic CD cases diagnosed in Spain. kDNA-qPCR showed higher sensitivity than Sat-qPCR and Sat-TcLAMP. According to all quantitative approaches, parasitemia levels were significantly higher in congenital infection than in chronic CD (1 × 10-1 to 5 × 105 versus >1 × 10-1 to 6 × 103 parasite equivalents/mL, respectively [P < 0.001]). Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR results were equivalent at high levels of parasitemia (P = 0.381). Discrepancies were significant for low levels of parasitemia and older individuals. Differences between Sat-TcLAMP and Sat-qPCR were not qualitatively significant, but estimations of parasitemia using Sat-TcLAMP were closer to those by kDNA-qPCR. Parasitemia changes were assessed in 6 individual cases in follow-up, in which trends showed similar patterns by all quantitative approaches. At high levels of parasitemia, Sat-TcLAMP, Sat-qPCR, and kDNA-qPCR worked similarly, but significant differences were found for the low levels characteristic of late chronic CD. A suitable harmonization strategy needs to be developed for low-level parasitemia detection using Sat-DNA- and kDNA-based tests. IMPORTANCE Currently, molecular equipment has been introduced into many health care centers, even in low-income countries. PCR, qPCR, and loop-mediated isothermal amplification (LAMP) are becoming more accessible for the diagnosis of neglected infectious diseases. Chagas disease (CD) is spreading worldwide, and in countries where the disease is not endemic, such as Spain, the parasite Trypanosoma cruzi is transmitted from mother to child (congenital CD). Here, we explore why LAMP, aimed at detecting T. cruzi parasite DNA, is a reliable option for the diagnosis of congenital CD and the early detection of reactivation in chronic infection. When the parasite load is high, LAMP is equivalent to any qPCR. In addition, the estimations of T. cruzi parasitemia in patients living in Spain, a country where the disease is not endemic, resemble natural evolution in areas of endemicity. If molecular tests are introduced into the diagnostic algorithm for congenital infection, early diagnosis and timely treatment would be accomplished, so the interruption of vertical transmission can be an achievable goal.

Simulating the Impacts of Augmenting Intensive Vector Control with Mass Drug Administration or Test-and-Treat Strategies on the Malaria Infectious Reservoir.

Highly effective vector control can reduce malaria burden significantly, but individuals with parasitemia provide a potential reservoir for onward transmission. We performed an empirical, non-parametric simulation based on cohort data from Tororo District, Uganda-an area with historically high but recently reduced malaria transmission-to estimate the effects of mass drug administration (MDA) and test-and-treat on parasite prevalence. We estimate that a single round of MDA would have accelerated declines in parasite prevalence dramatically over 2 years (cumulative parasite prevalence ratio [PPR], 0.34). This decline was mostly during the first year of administration (PPR, 0.23) and waned by 23 months (PPR, 0.74). Test-and-treat using a highly sensitive diagnostic had nearly the same effect as MDA at 1 year (PPR, 0.27) and required many fewer treatments. The impact of test-and-treat using a standard diagnostic was modest (PPR, 0.58 at 1 year). Our analysis suggests that in areas experiencing a dramatic reduction in malaria prevalence, MDA or test-and-treat with a highly sensitive diagnostic may be an effective way of reducing or eliminating the infectious reservoir temporarily. However, for sustained benefits, repeated rounds of the intervention or additional interventions are required.

Rapid diagnosis of Plasmodium falciparum malaria using a point-of-care loop-mediated isothermal amplification device.

LAMP diagnosis of malaria is simple and cost-effective with acceptable sensitivity and specificity as compared to standard diagnostic modules such as microscopy, RDTs and nested PCR, and thus its deployment for onsite screening of malaria in resource-limited regions is under consideration. However, the requirement of an electricity-operated dry bath and bulky read-out unit is still a major concern. In an effort to simplify this limitation, we have developed a portable LAMP device and fluorescence readout unit which can be used in the rapid point-of-care diagnosis of malaria. We have developed a point-of-care diagnostic LAMP device that is easy to operate by a mobile application, and the results can be quantified with a fluorescent readout unit. The diagnostic performance of the device was evaluated in 90 P. falciparum-infected clinical isolates stored at 4°C for 6-7 years and 10 freshly collected isolates from healthy volunteers. The LOD and quantitative ability of LAMP in estimating parasitemia levels were revealed with laboratory-grown P. falciparum strain (3D7). The LAMP assay performed in our device was exclusive for P. falciparum detection with sensitivity and specificity determined to be 98.89% and 100%, respectively, in clinical isolates. The LOD was documented to be 1 parasite/µl at the cut-off ADC value of 20. Parasite density estimated from ADC values showed concordance with microscopically determined parasite density of the cultured P. falciparum 3D7 strain. The LAMP assay performed in our device provides a possible portable platform for its deployment in the point-of-care diagnosis of malaria. Further validation of the quantitative ability of the assay with freshly collected or properly stored clinical samples of known parasitemia is necessary for field applicability.

Identification and Enumeration of Plasmodium falciparum Parasites by Light Microscopy.

Estimating malaria parasite density is important for patient care and management in rural and urban health centers in endemic areas. Here, we describe methodologically the protocols and methods to identify and enumerate Plasmodium falciparum parasites from infected blood using light microscopy. We provide step-by-step protocols and evaluate any possible drawbacks that may limit the methods and prospects of using light microscopy in diagnosing malaria.

Intermittent screening and treatment for malaria complementary to routine immunisation in the first year of life in Papua, Indonesia: a cluster randomised superiority trial.

BACKGROUND: In Papua (Indonesia), infants with P. falciparum and/or P. vivax malaria are at risk of severe anaemia and death. We hypothesized that in an area of high malaria transmission, intermittent screening and treatment of infants with malaria (ISTi) will reduce morbidity compared to passive case detection (PCDi). METHODS: We conducted a cluster randomised, open label, superiority trial. A total of 21 clusters of village health posts (VHP) were randomised 1:1 to either IST for infants coinciding with 4 routine immunisation visits or PCDi. Healthy term infants born to consenting mothers enrolled into a maternal malaria cluster randomised trial were included in the study and followed for 12 months. Point of care malaria rapid diagnostic tests were used to detect peripheral parasitaemia at 2, 3, 4 and 9 months old in all infants in ISTi clusters and when symptomatic in PCDi clusters. Infants with detected peripheral parasitaemia were treated with dihydroartemisinin-piperaquine. The co-primary outcomes were the incidence rate of clinical malaria in the first year of life and the prevalence of parasitaemia at age 12 months. The incidence rate ratio and prevalence ratio between ISTi and PCDi were estimated using mixed-effects Poisson and log-binomial regression modelling (accounting for clustering at VHP level). RESULTS: Between May 2014 and February 2017, 757 infants were enrolled into the study, 313 into 10 ISTi clusters, and 444 into 11 PCDi clusters. Overall, 132 episodes of parasitaemia were detected, of whom 17 (12.9%) were in symptomatic infants. Over 12 months, the incidence rate (IR) of clinical malaria was 24 [95% CI, 10-50] per 1000 children-years at risk in the ISTi arm and 19 [95% CI, 8,38] per 1000 children-years in the PCDi arm (adjusted incidence rate ratio [aIRR] 1.77 [95% CI, 0.62-5.01]; p = 0.280). The prevalence of parasitaemia at 12 months was 13% (33/254) in the IST clusters and 15% (57/379) in the PCD clusters (adjusted prevalence ratio (aPR) = 0.92 (95% CI, 0.70-1.21), p = 0.55). There was no difference in the risk of anaemia between treatment arms. CONCLUSIONS: In high malaria transmission area outside of Africa, our study suggests that compared to PCDi, ISTi offers no significant benefit in reducing the risk of clinical malaria in infants born to women receiving effective protection from malaria during pregnancy. TRIAL REGISTRATION: ClinicalTrials.gov NCT02001428 , registered on 20 Nov 2013.