Automated liquid handling robot for rapid lateral flow assay development.

The lateral flow assay (LFA) is one of the most popular technologies on the point-of-care diagnostics market due to its low cost and ease of use, with applications ranging from pregnancy to environmental toxins to infectious disease. While the use of these tests is relatively straightforward, significant development time and effort are required to create tests that are both sensitive and specific. Workflows to guide the LFA development process exist but moving from target selection to an LFA that is ready for field testing can be labor intensive, resource heavy, and time consuming. To reduce the cost and the duration of the LFA development process, we introduce a novel development platform centered on the flexibility, speed, and throughput of an automated robotic liquid handling system. The system comprises LFA-specific hardware and software that enable large optimization experiments with discrete and continuous variables such as antibody pair selection or reagent concentration. Initial validation of the platform was demonstrated during development of a malaria LFA but was readily expanded to encompass development of SARS-CoV-2 and Mycobacterium tuberculosis LFAs. The validity of the platform, where optimization experiments are run directly on LFAs rather than in solution, was based on a direct comparison between the robotic system and a more traditional ELISA-like method. By minimizing hands-on time, maximizing experiment size, and enabling improved reproducibility, the robotic system improved the quality and quantity of LFA assay development efforts.

Defining malaria risks among forest workers in Aceh, Indonesia: a formative assessment.

BACKGROUND: Following a dramatic decline of malaria cases in Aceh province, geographically-based reactive case detection (RACD) was recently evaluated as a tool to improve surveillance with the goal of malaria elimination. While RACD detected few cases in households surrounding index cases, engaging in forest work was identified as a risk factor for malaria and infections from Plasmodium knowlesi-a non-human primate malaria parasite-were more common than expected. This qualitative formative assessment was conducted to improve understanding of malaria risk from forest work and identify strategies for targeted surveillance among forest workers, including adapting reactive case detection. METHODS: Between June and August, 2016, five focus groups and 18 in-depth interviews with forest workers and key informants were conducted in each of four subdistricts in Aceh Besar and Aceh Jaya districts. Themes included: types of forest activities, mobility of workers, interactions with non-human primates, malaria prevention and treatment-seeking behaviours, and willingness to participate in malaria surveys at forest work sites and using peer-referral. RESULTS: Reported forest activities included mining, logging, and agriculture in the deep forest and along the forest fringe. Forest workers, particularly miners and loggers, described often spending weeks to months at work sites in makeshift housing, rarely utilizing mosquito prevention and, upon fever, self-medicating and seeking care from traditional healers or pharmacies rather than health facilities. Non-human primates are frequently observed near work sites, and most forest work locations are within a day's journey of health clinics. Employers and workers expressed interest in undertaking malaria testing and in participating in survey recruitment by peer-referral and at work sites. CONCLUSIONS: Diverse groups of forest workers in Aceh are potentially exposed to malaria through forest work. Passive surveillance and household-based screening may under-estimate malaria burden due to extended stays in the forest and health-seeking behaviours. Adapting active surveillance to specifically target forest workers through work-site screening and/or peer-referral appears promising for addressing currently undetected infections.

Cost-effectiveness analysis of malaria rapid diagnostic test in the elimination setting.

BACKGROUND: As more and more countries approaching the goal of malaria elimination, malaria rapid diagnostic tests (RDT) was recomendated to be a diagnostic strategy to achieve and maintain the statute of malaria free, as it's less requirments on equipment and experitise than microscopic examination. But there are very few economic evaluations to confirm whether RDT was cost-effective in the setting of malaria elimination. This research aimed to offer evidence for helping decision making on malaria diagnosis strategy. METHODS: A cost-effectiveness analysis was conducted to compare RDT with microscopy examination for malaria diagnosis, by using a decision tree model. There were three strategies of malaria diagnostic testing evaluated in the model, 1) microscopy, 2) RDT, 3) RDT followed by microscopy. The effect indicator was defined as the number of malaria cases treated appropriately. Based on the joint perspective of health sector and patient, costs data were collected from hospital information systems, key informant interviews, and patient surveys. Data collection was conducted in Jiangsu from September 2018 to January 2019. Epidemiological data were obtained from local malaria surveillance reports. A hypothetical cohort of 300 000 febrile patients were simulated to calculate the total cost and effect of each strategy. One-way, two-way, and probabilistic sensitivity analysis were performed to test the robustness of the result. RESULTS: The results showed that RDT strategy was the most effective (245 cases) but also the most costly (United States Dollar [USD] 4.47 million) compared to using microscopy alone (238 cases, USD 3.63 million), and RDT followed by microscopy (221 cases, USD 2.75 million). There was no strategy dominated. One-way sensitivity analysis reflected that the result was sensitive to the change in labor cost and two-way sensitivity analysis indicated that the result was not sensitive to the proportion of falciparum malaria. The result of Monte Carlo simulation showed that RDT strategy had higher effects and higher cost than other strategies with a high probability. CONCLUSIONS: Compared to microscopy and RDT followed by microscopy, RDT strategy had higher effects and higher cost in the setting of malaria elimination.

WHO malaria nucleic acid amplification test external quality assessment scheme: results of distribution programmes one to three.

BACKGROUND: The World Health Organization (WHO) recommends parasite-based diagnosis of malaria. In recent years, there has been surge in the use of various kinds of nucleic-acid amplification based tests (NAATs) for detection and identification of Plasmodium spp. to support clinical care in high-resource settings and clinical and epidemiological research worldwide. However, these tests are not without challenges, including lack (or limited use) of standards and lack of reproducibility, due in part to variation in protocols amongst laboratories. Therefore, there is a need for rigorous quality control, including a robust external quality assessment (EQA) scheme targeted towards malaria NAATs. To this effect, the WHO Global Malaria Programme worked with the UK National External Quality Assessment Scheme (UK NEQAS) Parasitology and with technical experts to launch a global NAAT EQA scheme in January 2017. METHODS: Panels of NAAT EQA specimens containing five major species of human-infecting Plasmodium at various parasite concentrations and negative samples were created in lyophilized blood (LB) and dried blood spot (DBS) formats. Two distributions per year were sent, containing five LB and five DBS specimens. Samples were tested and validated by six expert referee laboratories prior to distribution. Between 37 and 45 laboratories participated in each distribution and submitted results using the online submission portal of UK NEQAS. Participants were scored based on their laboratory's stated capacity to identify Plasmodium species, and individual laboratory reports were sent which included performance comparison with anonymized peers. RESULTS: Analysis of the first three distributions revealed that the factors that most significantly affected performance were sample format (DBS vs LB), species and parasite density, while laboratory location and the reported methodology used (type of nucleic acid extraction, amplification, or DNA vs RNA target) did not significantly affect performance. Referee laboratories performed better than non-referee laboratories. CONCLUSIONS: Globally, malaria NAAT assays now inform a range of clinical, epidemiological and research investigations. EQA schemes offer a way for laboratories to assess and improve their performance, which is critical to safeguarding the reliability of data and diagnoses especially in situations where various NAAT methodologies and protocols are in use.

Analysis on external competency assessment for malaria microscopists in China.

BACKGROUND: In order to meet the requirement of malaria elimination (ME), three courses of the External Competency Assessment of Malaria Microscopists (ECAMM) were conducted during 2017-2018 in China by facilitators designated by the World Health Organization (WHO-ECAMM). A training course with a model copied from the WHO-ECAMM course was also held a week ahead of ECAMM in March 2018. Thirty-six participants completed these courses and obtained different results. METHODS: The slide structures, agendas, score calculations, and the levels of certifications of the four courses strictly adhered to the WHO guidelines. All the data were collected in Excel 2016 and analysed in Graphpad Prism5 or SPSS 23. Significant differences were evaluated in Graphpad Prism5 by two-tailed paired t tests between the pre-assessment and final-assessment for each of the four courses, as well as one-way ANOVAs with Kruskal-Wallis tests and Dunn's post hoc tests among the final assessments of the four courses. Correlations between participants' competency results and their ages, years working on malaria, and numbers of malaria cases reported in their provinces were evaluated by bivariate correlations (two-tailed) and linear regression (excluding cases pairwise) in SPSS 23. The Pearson correlation coefficients (r values), P values (two tailed), adjusted R square (Adjusted R2), standardized coefficients (ß) and Sig. P values were recorded. The percentages of participants who gave the right answer to each slide (PPS) in the final assessments of the three WHO-ECAMM courses were calculated. Correlation analysis between PPS and parasitaemia (100-2000 parasites/µL) of Plasmodium falciparum slides used in species identification and parasite counting, were also evaluated via bivariate correlations (two-tailed) tests. RESULTS: Among the 36 participants, 16 participants were certificated as Level 1 (two from NRL), 10 were certified as Level 2 (one from NRL). Within the same course, participants had improved their average scores from pre-assessments to final assessments. The numbers of malaria cases reported in participants' provinces were strongly correlated to their species identification (SI) scores; r = 0.45, P = 0.040, n = 21; r = 0.57, P = 0.001, n = 32; r = 0.56, P = 0.007). The parasitaemia of P. falciparum within 100-2000 parasites/µL was correlated significantly (r = 0.44, P = 0.008, n = 36) with the PPS of all counting slides but not with slides for identification (r = - 0.018, P = 0.93, n = 30). CONCLUSIONS: The analysis and comparison of participants' competency results not only verified that the model of the WHO-ECAMM course had strong power in improving and assessing microscopists' competencies but also reflected the correlation between decreased numbers of indigenous malaria cases and microscopists' competencies in certain areas in China.

Increasing trends of malaria in a border area of the Greater Mekong Subregion.

BACKGROUND: Intensive malaria transmission along international borders is a significant impediment to malaria elimination in the Greater Mekong Subregion (GMS) of Southeast Asia. Passive case detection (PCD) was used to study the dynamics and trends of malaria transmission at the China-Myanmar border to provide epidemiologic information for improved malaria control. METHODS: PCD was conducted in one hospital and 12 clinics near the Laiza town in northeast Myanmar from 2011 to 2016. Clinical malaria was diagnosed by microscopy and demographic information was captured using a structured questionnaire at the time of the patient's presentation for care. RESULTS: Over the study period, 6175 (19.7%) malaria cases were confirmed by microscopy from 31,326 suspected cases. The four human malaria parasite species were all identified, with Plasmodium vivax and Plasmodium falciparum accounting for 5607 (90.8%) and 481 (7.8%) of the confirmed cases, respectively. In contrast to the steady decline of malaria in the general GMS, the study site had an upward trend of malaria incidence with vivax malaria outbreaks in 2013 and 2016. Adult males, children under the age of 15, and those with occupations such as farming, being a soldier or student, had significantly higher risks of clinical malaria compared to having fevers from other aetiologies. A self-reported history of clinical malaria was also associated with a higher risk of confirmed malaria. CONCLUSIONS: The China-Myanmar border area has experienced an overall upward trend of malaria incidence in recent years with P. vivax becoming the predominant species. Evidence-based control strategies need to focus on high-risk populations.

A Novel Assay for Simultaneous Assessment of Mammalian Host Blood, Mosquito Species, and Plasmodium spp. in the Medically Important Anopheles Mosquitoes of Madagascar.

Anopheles mosquitoes vary in habitat preference, feeding pattern, and susceptibility to various measures of vector control. Consequently, it is important that we identify reservoirs of disease, identify vectors, and characterize feeding patterns to effectively implement targeted control measures. Using 467 anopheline mosquito abdomen squashes captured in Madagascar, we designed a novel ligase detection reaction and fluorescent microsphere assay, dubbed Bloodmeal Detection Assay for Regional Transmission (BLOODART), to query the bloodmeal content, identify five Anopheles mosquito species, and detect Plasmodium infection. Validation of mammalian bloodspots was achieved by preparation and analysis of known hosts (singular and mixed), sensitivity to degradation and storage method were assessed through mosquito feeding experiments, and quantification was explored by altering ratios of two mammal hosts. BLOODART identifications were validated by comparison with mosquito samples identified by sequenced portions of the internal transcribed spacer 2. BLOODART identification of control mammal bloodspots was 100% concordant for singular and mixed mammalian blood. BLOODART was able to detect hosts up to 42 hours after digestion when mosquito samples were stored in ethanol. A mammalian host was identified in every field-collected, blood-fed female Anopheles mosquito by BLOODART. The predominant mosquito host was cow (n = 451), followed by pig (n = 26) and human (n = 25). Mixed species bloodmeals were commonly observed (n = 33). A BLOODART molecular identification was successful for 318/467 mosquitoes, with an overall concordance of 60% with all field-captured, morphologically identified Anopheles specimens. BLOODART enables characterization of large samples and simultaneous pathogen detection to monitor and incriminate disease vectors in Madagascar.

A fast and cost-effective microsampling protocol incorporating reduced animal usage for time-series transcriptomics in rodent malaria parasites.

BACKGROUND: The transcriptional regulation that occurs in malaria parasites during the erythrocytic stages of infection can be studied in vivo with rodent malaria parasites propagated in mice. Time-series transcriptome profiling commonly involves the euthanasia of groups of mice at specific time points followed by the extraction of parasite RNA from whole blood samples. Current methodologies for parasite RNA extraction involve several steps and when multiple time points are profiled, these protocols are laborious, time-consuming, and require the euthanization of large cohorts of mice. RESULTS: A simplified protocol has been designed for parasite RNA extraction from blood volumes as low as 20 µL (microsamples), serially bled from mice via tail snips and directly lysed with TRIzol reagent. Gene expression data derived from microsampling using RNA-seq were closely matched to those derived from larger volumes of leucocyte-depleted and saponin-treated blood obtained from euthanized mice with high reproducibility between biological replicates. Transcriptome profiling of microsamples taken at different time points during the intra-erythrocytic developmental cycle of the rodent malaria parasite Plasmodium vinckei revealed the transcriptional cascade commonly observed in malaria parasites. CONCLUSIONS: Microsampling is a quick, robust and cost-efficient approach to sample collection for in vivo time-series transcriptomic studies in rodent malaria parasites.

Comparative Assessment of Diagnostic Performances of Two Commercial Rapid Diagnostic Test Kits for Detection of Plasmodium spp. in Ugandan Patients with Malaria.

Prompt diagnosis of malaria cases with rapid diagnostic tests (RDTs) has been widely adopted as an effective malaria diagnostic tool in many malaria endemic countries, primarily due to their easy operation, fast result output, and straightforward interpretation. However, there has been controversy about the diagnostic accuracy of RDTs. This study was conducted to evaluate the diagnostic performances of the 2 commercially available malaria RDT kits, RapiGEN Malaria Ag Pf/Pv (pLDH/pLDH) and Asan EasyTestTM Malaria Ag Pf/Pv (HRP-2/pLDH) for their abilities to detect Plasmodium species in blood samples collected from Ugandan patients with malaria. To evaluate the diagnostic performances of these 2 RDT kits, 229 blood samples were tested for malaria infection by microscopic examination and a species-specific nested polymerase chain reaction. The detection sensitivities for P. falciparum of Malaria Ag Pf/Pv (pLDH/pLDH) and Asan EasyTestTM Malaria Ag Pf/Pv (HRP-2/pLDH) were 87.83% and 89.57%, respectively. The specificities of the 2 RDTs were 100% for P. falciparum and mixed P. falciparum/P. vivax infections. These results suggest that the 2 RDT kits showed reasonable levels of diagnostic performances for detection of the malaria parasites from Ugandan patients. However, neither kit could effectively detect P. falciparum infections with low parasitaemia (<500 parasites/µl).

Costs and cost-effectiveness of malaria reactive case detection using loop-mediated isothermal amplification compared to microscopy in the low transmission setting of Aceh Province, Indonesia.

BACKGROUND: Reactive case detection (RACD) is an active case finding strategy where households and neighbours of a passively identified case (index case) are screened to identify and treat additional malaria infections with the goal of gathering surveillance information and potentially reducing further transmission. Although it is widely considered a key strategy in low burden settings, little is known about the costs and the cost-effectiveness of different diagnostic methods used for RACD. The aims of this study were to measure the cost of conducting RACD and compare the cost-effectiveness of microscopy to the more sensitive diagnostic method loop-mediated isothermal amplification (LAMP). METHODS: The study was conducted in RACD surveillance sites in five sub-districts in Aceh Besar, Indonesia. The cost inputs and yield of implementing RACD with microscopy and/or LAMP were collected prospectively over a 20 months study period between May 2014 and December 2015. Costs and cost-effectiveness (USD) of the different strategies were examined. The main cost measures were cost per RACD event, per person screened, per population at risk (PAR); defined as total population in each sub-district, and per infection found. The main cost-effectiveness measure was incremental cost-effectiveness ratio (ICER), expressed as cost per malaria infection detected by LAMP versus microscopy. The effects of varying test positivity rate or diagnostic yield on cost per infection identified and ICER were also assessed. RESULTS: Among 1495 household members and neighbours screened in 36 RACD events, two infections were detected by microscopy and confirmed by LAMP, and four infections were missed by microscopy but detected by LAMP. The average total cost of conducting RACD using microscopy and LAMP was $1178 per event with LAMP-specific consumables and personnel being the main cost drivers. The average cost of screening one individual during RACD was $11, with an additional cost of diagnostics at $0.62 and $16 per person for microscopy and LAMP, respectively. As a public health intervention, RACD using both diagnostics cost an average of $0.42 per PAR per year. Comparing RACD using microscopy only versus RACD using LAMP only, the cost per infection found was $8930 and $6915, respectively. To add LAMP as an additional intervention accompanying RACD would cost $9 per individual screened annually in this setting. The ICER was estimated to be $5907 per additional malaria infection detected by LAMP versus microscopy. Cost per infection identified and ICER declined with increasing test positivity rate and increasing diagnostic yield. CONCLUSIONS: This study provides the first estimates on the cost and cost-effectiveness of RACD from a low transmission setting. Costs per individual screened were high, though costs per PAR were low. Compared to microscopy, the use of LAMP in RACD was more costly but more cost-effective for the detection of infections, with diminishing returns observed when findings were extrapolated to scenarios with higher prevalence of infection using more sensitive diagnostics. As malaria programmes consider active case detection and the integration of more sensitive diagnostics, these findings may inform strategic and budgetary planning.

An assessment of mosquito collection techniques for xenomonitoring of anopheline-transmitted Lymphatic Filariasis in Ghana.

Monitoring vectors is relevant to ascertain transmission of lymphatic filariasis (LF). This may require the best sampling method that can capture high numbers of specific species to give indication of transmission. Gravid anophelines are good indicators for assessing transmission due to close contact with humans through blood meals. This study compared the efficiency of an Anopheles gravid trap (AGT) with other mosquito collection methods including the box and the Centres for Disease Control and Prevention gravid, light, exit and BioGent-sentinel traps, indoor resting collection (IRC) and pyrethrum spray catches across two endemic regions of Ghana. The AGT showed high trapping efficiency by collecting the highest mean number of anophelines per night in the Western (4.6) and Northern (7.3) regions compared with the outdoor collection methods. Additionally, IRC was similarly efficient in the Northern region (8.9) where vectors exhibit a high degree of endophily. AGT also showed good trapping potential for collecting Anopheles melas which is usually difficult to catch with existing methods. Screening of mosquitoes for infection showed a 0.80-3.01% Wuchereria bancrofti and 2.15-3.27% Plasmodium spp. in Anopheles gambiae. The AGT has shown to be appropriate for surveying Anopheles populations and can be useful for xenomonitoring for both LF and malaria.

External quality assessment of malaria microscopy diagnosis in selected health facilities in Western Oromia, Ethiopia.

BACKGROUND: Accurate early diagnosis and prompt treatment are one of the key strategies to control and prevent malaria disease. External quality assessment is the most effective method for evaluation of the quality of malaria microscopy diagnosis. The aim of this study was to assess the quality of malaria microscopy diagnosis and its associated factors in selected public health facility laboratories in East Wollega Zone, Western Ethiopia. METHODS: Facility-based cross-sectional study design was conducted in 30 randomly selected public health facility laboratories from November 2014 to January 2015 in East Wollega Zone, Western Ethiopia. Ten validated stained malaria panel slides with known Plasmodium species, developmental stage and parasite density were distributed. Data were captured; cleaned and analyzed using SPSS version 20 statistical software-multivariate logistic regressions and the agreement in reading between the peripheral diagnostic centers and the reference laboratory were done using kappa statistics. RESULTS: A total of 30 health facility laboratories were involved in the study and the overall quality of malaria microscopy diagnosis was poor (62.3%). The associated predictors of quality in this diagnosis were in-service training [(AOR = 16, 95% CI (1.3, 1.96)], smearing quality [(AOR = 24, 95% CI (1.8, 3.13)], staining quality [(AOR = 15, 95% CI (2.35, 8.61), parasite detection [(AOR = 9, 95% CI (1.1, 8.52)] and identification skills [(AOR = 8.6, 95% CI (1.21, 1.63)]. Eighteen (60%) of health facility laboratories had in-service trained laboratory professionals on malaria microscopy diagnosis. CONCLUSION: Overall quality of malaria microscopy diagnosis was poor and a significant gap in this service was observed that could impact on its diagnostic services.

Rapid Diagnostic for Point-of-Care Malaria Screening.

Despite significant success in therapeutic development, malaria remains a widespread and deadly infectious disease in the developing world. Given the nearly 100% efficacy of current malaria therapeutics, the primary barrier to eradication is lack of early diagnosis of the infected population. However, there are multiple strains of malaria. Although significant efforts and resources have been invested in developing antibody-based diagnostic methods for Plasmodium falciparum, a rapid and easy to use screening method capable of detecting all malaria strains has not been realized. Yet, until the entire malaria-infected population receives treatment, the disease will continue to impact society. Here, we report the development of a portable, magneto-optic technology for early stage malaria diagnosis based on the detection of the malaria pigment, hemozoin. Using ß-hematin, a hemozoin mimic, we demonstrate detection limits of <0.0081 µg/mL in 500 µL of whole rabbit blood with no additional reagents required. This level corresponds to <26 parasites/µL, a full order of magnitude below clinical relevance and comparable to or less than existing technologies.

Assessment of asymptomatic Plasmodium spp. infection by detection of parasite DNA in residents of an extra-Amazonian region of Brazil.

BACKGROUND: The hypotheses put forward to explain the malaria transmission cycle in extra-Amazonian Brazil, an area of very low malaria incidence, are based on either a zoonotic scenario involving simian malaria, or a scenario in which asymptomatic carriers play an important role. OBJECTIVES: To determine the incidence of asymptomatic infection by detecting Plasmodium spp. DNA and its role in residual malaria transmission in a non-Amazonian region of Brazil. METHODS: Upon the report of the first malaria case in 2010 in the Atlantic Forest region of the state of Espírito Santo, inhabitants within a 2 km radius were invited to participate in a follow-up study. After providing signed informed consent forms, inhabitants filled out a questionnaire and gave blood samples for PCR, and thick and thin smears. Follow-up visits were performed every 3 months over a 21 month period, when new samples were collected and information was updated. RESULTS: Ninety-two individuals were initially included for follow-up. At the first collection, all of them were clearly asymptomatic. One individual was positive for Plasmodium vivax, one for Plasmodium malariae and one for both P. vivax and P. malariae, corresponding to a prevalence of 3.4% (2.3% for each species). During follow-up, four new PCR-positive cases (two for each species) were recorded, corresponding to an incidence of 2.5 infections per 100 person-years or 1.25 infections per 100 person-years for each species. A mathematical transmission model was applied, using a low frequency of human carriers and the vector density in the region, and calculated based on previous studies in the same locality whose results were subjected to a linear regression. This analysis suggests that the transmission chain is unlikely to be based solely on human carriers, regardless of whether they are symptomatic or not. CONCLUSION: The low incidence of cases and the low frequency of asymptomatic malaria carriers investigated make it unlikely that the transmission chain in the region is based solely on human hosts, as cases are isolated one from another by hundreds of kilometers and frequently by long periods of time, reinforcing instead the hypothesis of zoonotic transmission.

Malaria in Brazil, Colombia, Peru and Venezuela: current challenges in malaria control and elimination.

In spite of significant progress towards malaria control and elimination achieved in South America in the 2000s, this mosquito-transmitted tropical disease remains an important public health concern in the region. Most malaria cases in South America come from Amazon rain forest areas in northern countries, where more than half of malaria is caused by Plasmodium vivax, while Plasmodium falciparum malaria incidence has decreased in recent years. This review discusses current malaria data, policies and challenges in four South American Amazon countries: Brazil, Colombia, Peru and the Bolivarian Republic of Venezuela. Challenges to continuing efforts to further decrease malaria incidence in this region include: a significant increase in malaria cases in recent years in Venezuela, evidence of submicroscopic and asymptomatic infections, peri-urban malaria, gold mining-related malaria, malaria in pregnancy, glucose-6-phosphate dehydrogenase (G6PD) deficiency and primaquine use, and possible under-detection of Plasmodium malariae. Some of these challenges underscore the need to implement appropriate tools and procedures in specific regions, such as a field-compatible molecular malaria test, a P. malariae-specific test, malaria diagnosis and appropriate treatment as part of regular antenatal care visits, G6PD test before primaquine administration for P. vivax cases (with weekly primaquine regimen for G6PD deficient individuals), single low dose of primaquine for P. falciparum malaria in Colombia, and national and regional efforts to contain malaria spread in Venezuela urgently needed especially in mining areas. Joint efforts and commitment towards malaria control and elimination should be strategized based on examples of successful regional malaria fighting initiatives, such as PAMAFRO and RAVREDA/AMI.

Markov chain Monte Carlo and expectation maximization approaches for estimation of haplotype frequencies for multiply infected human blood samples.

BACKGROUND: Haplotypes are important in anti-malarial drug resistance because genes encoding drug resistance may accumulate mutations at several codons in the same gene, each mutation increasing the level of drug resistance and, possibly, reducing the metabolic costs of previous mutation. Patients often have two or more haplotypes in their blood sample which may make it impossible to identify exactly which haplotypes they carry, and hence to measure the type and frequency of resistant haplotypes in the malaria population. RESULTS: This study presents two novel statistical methods expectation-maximization (EM) and Markov chain Monte Carlo (MCMC) algorithms to investigate this issue. The performance of the algorithms is evaluated on simulated datasets consisting of patient blood characterized by their multiplicity of infection (MOI) and malaria genotype. The datasets are generated using different resistance allele frequencies (RAF) at each single nucleotide polymorphisms (SNPs) and different limit of detection (LoD) of the SNPs and the MOI. The EM and the MCMC algorithm are validated and appear more accurate, faster and slightly less affected by LoD of the SNPs and the MOI compared to previous related statistical approaches. CONCLUSIONS: The EM and the MCMC algorithms perform well when analysing malaria genetic data obtained from infected human blood samples. The results are robust to genotyping errors caused by LoDs and function well even in the absence of MOI data on individual patients.

Comparative assessment of genomic DNA extraction processes for Plasmodium: Identifying the appropriate method.

Plasmodium DNA, in addition to being used for molecular diagnosis of malaria, find utility in monitoring patient responses to antimalarial drugs, drug resistance studies, genotyping and sequencing purposes. Over the years, numerous protocols have been proposed for extracting Plasmodium DNA from a variety of sources. Given that DNA isolation is fundamental to successful molecular studies, here we review the most commonly used methods for Plasmodium genomic DNA isolation, emphasizing their pros and cons. A comparison of these existing methods has been made, to evaluate their appropriateness for use in different applications and identify the method suitable for a particular laboratory based study. Selection of a suitable and accessible DNA extraction method for Plasmodium requires consideration of many factors, the most important being sensitivity, cost-effectiveness and, purity and stability of isolated DNA. Need of the hour is to accentuate on the development of a method that upholds well on all these parameters.

Capture and Ligation Probe-PCR (CLIP-PCR) for Molecular Screening, with Application to Active Malaria Surveillance for Elimination.

BACKGROUND: Malaria control programs have achieved remarkable success during the past decade. Nonetheless, sensitive and affordable methods for active screening of malaria parasites in low-transmission settings remain urgently needed. METHODS: We developed a molecular screening method, capture and ligation probe-PCR (CLIP-PCR), which achieved the sensitivity of reverse-transcription PCR but eliminated the reliance on RNA purification and reverse transcription. In this method, 18S rRNA of genus Plasmodium is released from blood, captured onto 96-well plates, and quantified by the amount of ligated probes that bind continuously to it. We first used laboratory-prepared samples to test the method across a range of parasite densities and pool sizes, then applied the method to an active screening of 3358 dried blood spot samples collected from 3 low-endemic areas in China. RESULTS: Plasmodium falciparum diluted in whole blood lysate could be detected at a concentration as low as 0.01 parasites/µL, and a pool size of ≤36 did not significantly affect assay performance. When coupled with a matrix pooling strategy, the assay drastically increased throughput to thousands of samples per run while reducing the assay cost to cents per sample. In the active screening, CLIP-PCR identified 14 infections, including 4 asymptomatic ones, with <500 tests, costing

SMS photograph-based external quality assessment of reading and interpretation of malaria rapid diagnostic tests in the Democratic Republic of the Congo.

BACKGROUND: The present External Quality Assessment (EQA) assessed reading and interpretation of malaria rapid diagnostic tests (RDTs) in the Democratic Republic of the Congo (DRC). METHODS: The EQA consisted of (i) 10 high-resolution printed photographs displaying cassettes with real-life results and multiple choice questions (MCQ) addressing individual health workers (HW), and (ii) a questionnaire on RDT use addressing the laboratory of health facilities (HF). Answers were transmitted through short message services (SMS). RESULTS: The EQA comprised 2344 HW and 1028 HF covering 10/11 provinces in DRC. Overall, median HW score (sum of correct answers on 10 MCQ photographs for each HW) was 9.0 (interquartile range 7.5 - 10); MCQ scores (the % of correct answers for a particular photograph) ranged from 54.8% to 91.6%. Most common errors were (i) reading or interpreting faint or weak line intensities as negative (3.3%, 7.2%, 24.3% and 29.1% for 4 MCQ photographs), (ii) failure to distinguish the correct Plasmodium species (3.4% to 7.0%), (iii) missing invalid test results (8.4% and 23.6%) and (iv) missing negative test results (10.0% and 12.4%). HW who were trained less than 12 months ago had best MCQ scores for 7/10 photographs as well as a significantly higher proportion of 10/10 scores, but absolute differences in MCQ scores were small. HW who had participated in a previous EQA performed significantly better for 4/10 photographs compared to those who had not. Except for two photographs, MCQ scores were comparable for all levels of the HF hierarchy and non-laboratory staff (HW from health posts) had similar performance as to laboratory staff. Main findings of the questionnaire were (i) use of other RDT products than recommended by the national malaria control programme (nearly 20% of participating HF), (ii) lack of training for a third (33.6%) of HF, (iii) high proportions (two-thirds, 66.5%) of HF reporting stock-outs. CONCLUSIONS: The present EQA revealed common errors in RDT reading and interpretation by HW in DRC. Performances of non-laboratory and laboratory staff were similar and dedicated training was shown to improve HW competence although to a moderate extent. Problems in supply, distribution and training of RDTs were detected.

Towards malaria microscopy at the point-of-contact: an assessment of the diagnostic performance of the Newton Nm1 microscope in Uganda.

Malaria microscopy in sub-Saharan Africa is often restricted by access to light microscopes. To address this gap, a novel portable inverted monocular microscope, the Newton Nm1, was designed and is now commercially available. Its diagnostic performance was assessed in a blinded-slide trial at ×1000 (oil) of Giemsa-stained thick blood films against a conventional microscope as undertaken by four Ugandan Ministry of Health technicians. With the Newton Nm1, diagnostic performance was: sensitivity 93.5% (95% confidence interval (CI) 78.6-99.2%), specificity 100.0% (95% CI 82.4-100.0%), positive predictive value 100.0% (95% CI 88.1-100.0%) and negative predictive value 90.5% (95% CI 69.6-98.8%). Discordance was due to a systematic error underestimating parasitaemia by ~45%; when counting Plasmodium parasites against 200 white blood cells, blood films with low parasitaemia (i.e. <100 µL(-1) of blood) could be overlooked and misclassified. By contrast, specificity was excellent with no false positives encountered. Whilst proven useful, especially in resource-poor environments, it is still unclear how we can ensure the uptake of the Newton Nm1 within sub-Saharan Africa.