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.

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%.

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.

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.

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.

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.

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.

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.

Evaluation of two commercial kits and two laboratory-developed qPCR assays compared to LAMP for molecular diagnosis of malaria.

BACKGROUND: Malaria is an infectious disease considered as one of the biggest causes of mortality in endemic areas. This life-threatening disease needs to be quickly diagnosed and treated. The standard diagnostic tools recommended by the World Health Organization are thick blood smears microscopy and immuno-chromatographic rapid diagnostic tests. However, these methods lack sensitivity especially in cases of low parasitaemia and non-falciparum infections. Therefore, the need for more accurate and reliable diagnostic tools, such as real-time polymerase chain reaction based methods which have proven greater sensitivity particularly in the screening of malaria, is prominent. This study was conducted at the French National Malaria Reference Centre to assess sensitivity and specificity of two commercial malaria qPCR kits and two in-house developed qPCRs compared to LAMP. METHODS: 183 blood samples received for expertise at the FNMRC were included in this study and were subjected to four different qPCR methods: the Biosynex Ampliquick® Malaria test, the BioEvolution Plasmodium Typage test, the in-house HRM and the in-house TaqMan qPCRs. The specificity and sensitivity of each method and their confidence intervals were determined with the LAMP-based assay Alethia® Malaria as the reference for malaria diagnosis. The accuracy of species diagnosis of the Ampliquick® Malaria test and the two in-house qPCRs was also evaluated using the BioEvolution Plasmodium Typage test as the reference method for species identification. RESULTS: The main results showed that when compared to LAMP, a test with excellent diagnostic performances, the two in-house developed qPCRs were the most sensitive (sensitivity at 100% for the in-house TaqMan qPCR and 98.1% for the in-house HRM qPCR), followed by the two commercial kits: the Biosynex Ampliquick® Malaria test (sensitivity at 97.2%) and the BioEvolution Plasmodium Typage (sensitivity at 95.4%). Additionally, with the in-house qPCRs we were able to confirm a Plasmodium falciparum infection in microscopically negative samples that were not detected by commercial qPCR kits. This demonstrates that the var genes of P. falciparum used in these in-house qPCRs are more reliable targets than the 18S sRNA commonly used in most of the developed qPCR methods for malaria diagnosis. CONCLUSION: Overall, these results accentuate the role molecular methods could play in the screening of malaria. This may represent a helpful tool for other laboratories looking to implement molecular diagnosis methods in their routine analysis, which could be essential for the detection and treatment of malaria carriers and even for the eradication of this disease.

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.

High Asymptomatic Cases of Babesiosis in Dogs and Comparison of Diagnostic Performance of Conventional PCR vs Blood Smears.

PURPOSE: Babesiosis is one of the most important globally extended and quickly spreading tick-borne infections of dogs. Diagnosis of babesiosis in Sri Lanka is based on clinical signs followed by thin blood smears which could be error-prone due to undetected early infections, absence of clinical signs or low parasitemia. The present study investigated the prevalence of babesiosis in dogs presented to the Veterinary Teaching Hospital (VTH) at the Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, for treatments, vaccinations, and regular check-ups, and compared the diagnosis methods of microscopy and molecular analysis. METHODS: Blood samples from dogs were collected from January to June 2019. First, Giemsa stained blood smears were prepared, and then the blood samples were subjected to PCR using genus-specific primers to amplify a 411-450 bp region in the 18S rRNA gene. Twenty samples from PCR amplified products were sequenced for species identification and phylogenetic analysis. Clinical signs of the dogs were noted down, and ticks were also collected from dogs if any. RESULTS: Results show a very high prevalence of canine babesiosis (78.6%) among the dogs brought to the VTH. The parasite was identified microscopically and genetically as Babesia gibsoni. A large percentage (66.7%) of infections was asymptomatic. Out of 42 blood samples, 19 (45.2%) were microscopically positive for babesiosis while 33 (78.6%) were PCR positive, showing a significant difference in the two methods of diagnosis (chi-square test, χ2 = 9.462, p = 0.002). Three tick species: Rhipicephalus haemaphysaloides, Haemaphysalis bispinosa, and Rhipicephalus sanguineus were found attached to the dogs. CONCLUSION: This study shows a very high prevalence of canine babesiosis among dogs in the Kandy area. Most of these infections might go undetected if only microscopy was used to diagnose. An improved, rapid diagnostic method such as the novel, PCR-based point-of-care diagnostic method that detects very low parasitemia within 30 min is needed. Moreover, as most infected dogs did not show clinical signs, they may act as reservoirs of infection. The ability of asymptomatic dogs to spread babesiosis should be investigated.

Field evaluation of the diagnostic performance of EasyScan GO: a digital malaria microscopy device based on machine-learning.

BACKGROUND: Microscopic examination of Giemsa-stained blood films remains the reference standard for malaria parasite detection and quantification, but is undermined by difficulties in ensuring high-quality manual reading and inter-reader reliability. Automated parasite detection and quantification may address this issue. METHODS: A multi-centre, observational study was conducted during 2018 and 2019 at 11 sites to assess the performance of the EasyScan Go, a microscopy device employing machine-learning-based image analysis. Sensitivity, specificity, accuracy of species detection and parasite density estimation were assessed with expert microscopy as the reference. Intra- and inter-device reliability of the device was also evaluated by comparing results from repeat reads on the same and two different devices. This study has been reported in accordance with the Standards for Reporting Diagnostic accuracy studies (STARD) checklist. RESULTS: In total, 2250 Giemsa-stained blood films were prepared and read independently by expert microscopists and the EasyScan Go device. The diagnostic sensitivity of EasyScan Go was 91.1% (95% CI 88.9-92.7), and specificity 75.6% (95% CI 73.1-78.0). With good quality slides sensitivity was similar (89.1%, 95%CI 86.2-91.5), but specificity increased to 85.1% (95%CI 82.6-87.4). Sensitivity increased with parasitaemia rising from 57% at < 200 parasite/µL, to ≥ 90% at > 200-200,000 parasite/µL. Species were identified accurately in 93% of Plasmodium falciparum samples (kappa = 0.76, 95% CI 0.69-0.83), and in 92% of Plasmodium vivax samples (kappa = 0.73, 95% CI 0.66-0.80). Parasite density estimates by the EasyScan Go were within ± 25% of the microscopic reference counts in 23% of slides. CONCLUSIONS: The performance of the EasyScan Go in parasite detection and species identification accuracy fulfil WHO-TDR Research Malaria Microscopy competence level 2 criteria. In terms of parasite quantification and false positive rate, it meets the level 4 WHO-TDR Research Malaria Microscopy criteria. All performance parameters were significantly affected by slide quality. Further software improvement is required to improve sensitivity at low parasitaemia and parasite density estimations. Trial registration ClinicalTrials.gov number NCT03512678.