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Background The emergence and spread of Plasmodium falciparum parasites that lack HRP2/3 proteins and the resulting decreased utility of HRP2-based malaria rapid diagnostic tests (RDTs) prompted the World Health Organization and other global health stakeholders to prioritize the discovery of novel diagnostic biomarkers for malaria. Methods To address this pressing need, we adopted a dual, systematic approach by conducting a systematic review of the literature for publications on diagnostic biomarkers for uncomplicated malaria and a systematic in silico analysis of P. falciparum proteomics data for Plasmodium proteins with favorable diagnostic features. Results Our complementary analyses led us to 2 novel malaria diagnostic biomarkers compatible for use in an RDT format: glyceraldehyde 3-phosphate dehydrogenase and dihydrofolate reductase-thymidylate synthase. Conclusions Overall, our results pave the way for the development of next-generation malaria RDTs based on new antigens by identifying 2 lead candidates with favorable diagnostic features and partially de-risked product development prospects., The World Health Organization called for the identification of novel biomarkers that can fill the diagnostic gap in settings where hrp2/3 deletions are common. By adopting a dual approach, we identified 2 candidates, glyceraldehyde 3-phosphate dehydrogenase and dihydrofolate reductase-thymidylate synthase, with favorable diagnostic features.

Plasmodium vivax is co-endemic with Plasmodium falciparum in Peru, and optimum management requires distinguishing these two species in the blood of patients. For the differential identification of P. vivax and other Plasmodium spp., the LoopampTM Malaria Pan Detection Kit in combination with the Loopamp Malaria Pv Detection Kit (Eiken Chemical Co. Ltd., Tokyo, Japan) was used to evaluate 559 whole blood samples collected in 2017 from febrile patients with suspected malaria attending different health facilities in the Loreto region. The Loopamp Malaria Pan Detection Kit showed a sensitivity of 87.7% (95% CI: 83.5-91.9) and a specificity of 94.4% (95% CI: 91.9-96.9) and good agreement with PCR (Cohen's kappa 0.8266, 95% CI: 0.7792-0.874). By comparison, the Loopamp Malaria Pv Detection Kit showed a similar sensitivity (84.4%, 95% CI: 79.0-89.7) and specificity (92.4%, 95% CI: 89.7-95.0) and substantial agreement with PCR (Cohen's kappa: 0.7661, 95% CI: 0.7088-0.8234).

Background In low transmission settings early diagnosis is the main strategy to reduce adverse outcomes of malaria in pregnancy; however, microscopy and rapid diagnostic tests (RDTs) are inadequate for detecting low-density infections. We studied the performance of the highly sensitive-RDT (hsRDT) and the loop mediated isothermal DNA amplification (LAMP) for the detection of P. falciparum in pregnant women. Methods A cross-sectional study was conducted in two malaria-endemic municipalities in Colombia. We screened pregnant women in the context of an antenatal care program in health facilities and evaluated five tests (microscopy, conventional RDT, hsRDT, LAMP and nested polymerase chain reaction-PCR) for the detection of P. falciparum in peripheral blood, using a quantitative reverse transcription PCR (qRT-PCR) as the reference standard. Diagnostic performance of hsRDT and LAMP were compared with routine testing. Results The prevalence of P. falciparum was 4.5% by qRT-PCR, half of those infections were subpatent. The sensitivity of the hsRDT (64.1%) was slightly better compared to microscopy and cRDT (59 and 53.8% respectively). LAMP had the highest sensitivity (89.7%) for detecting P. falciparum and the ability to detect very low-density infections (minimum parasite density detected 0.08 p/μL). Conclusions There is an underestimation of Plasmodium spp. infections by tests routinely used in pregnant women attending antenatal care visits. LAMP methodology can be successfully implemented at local hospitals in malaria-endemic areas. The relevance of detecting and treating this sub-patent P. falciparum infections in pregnant women should be evaluated. Trial registration ClinicalTrials.gov, Identifier: NCT03172221, Date of registration: May 29, 2017.

Background Highly sensitive and accurate malaria diagnostic tools are essential to identify asymptomatic low parasitaemia infections. This study evaluated the performance of histidine-rich protein 2 (HRP-2) based rapid diagnostic tests (RDTs), microscopy and loop-mediated isothermal amplification (LAMP) for the detection of asymptomatic Plasmodium spp. infections in Northern Côte d’Ivoire, using nested polymerase chain reaction (nPCR) as reference. Methods A household-based survey was carried out in July 2016, in the health district of Korhogo, involving 1011 adults without malaria symptom nor history of fever during the week before recruitment. The fresh capillary blood samples were collected to detect Plasmodium infections using on HRP-2-based RDTs, microscopy and LAMP and stored as dried blood spots (DBS). A subset of the DBS (247/1011, 24.4%) was randomly selected for nPCR analyses. Additionally, venous blood samples, according to LAMP result (45 LAMP positive and 65 LAMP negative) were collected among the included participants to perform the nested PCR used as the reference. Results The prevalence of asymptomatic Plasmodium spp. infections determined by RDT, microscopy, and LAMP were 4% (95% confidence interval (CI) 2.8–5.3), 5.2% (95% CI 3.9–6.6) and 18.8% (95% CI 16.4–21.2), respectively. Considering PCR on venous blood as reference, performed on 110 samples, the sensibility and specificity were, respectively, 17.8% (95% CI 6.1–29.4) and 100% for RDT, 20.0% (95% CI 7.8–32) and 100% for microscopy, and 93.3% (95% CI 85.7–100) and 95.4% (95% CI 92.2–100) for LAMP. Conclusion In Northern Côte d’Ivoire, asymptomatic Plasmodium infection was found to be widely distributed as approximately one out of five study participants was found to be Plasmodium infected. LAMP appears currently to be the only available diagnostic method that can identify in the field this reservoir of infections and should be the method to consider for potential future active case detection interventions targeting elimination of these infections.

Background The radical cure of Plasmodium vivax requires treatment with an 8-aminoquinoline drug, such as primaquine and tafenoquine, to eradicate liver hypnozoite stages, which can reactivate to cause relapsing infections. Safe treatment regimens require prior screening of patients for glucose-6-phosphate dehydrogenase (G6PD) deficiency to avoid potential life-threatening drug induced haemolysis. Testing is rarely available in malaria endemic countries, but will be needed to support routine use of radical cure. This study investigates end-user perspectives in Bangladesh on the introduction of a quantitative G6PD test (SD Biosensor STANDARD™ G6PD analyser) to support malaria elimination. Methods The perspectives of users on the SD Biosensor test were analysed using semi-structured interviews and focus group discussions with health care providers and malaria programme officers in Bangladesh. Key emerging themes regarding the feasibility of introducing this test into routine practice, including perceived barriers, were analysed. Results In total 63 participants were interviewed. Participants emphasized the life-saving potential of the biosensor, but raised concerns including the impact of limited staff time, high workload and some technical aspects of the device. Participants highlighted that there are both too few and too many P. vivax patients to implement G6PD testing owing to challenges of funding, workload and complex testing infrastructure. Implementing the biosensor would require flexibility and improvisation to deal with remote sites, overcoming a low index of suspicion and mutual interplay of declining patient numbers and reluctance to test. This approach would generate new forms of evidence to justify introduction in policy and carefully consider questions of deployment given declining patient numbers. Conclusions The results of the study show that, in an elimination context, the importance of malaria needs to be maintained for both policy makers and the affected communities, in this case by ensuring P. vivax, PQ treatment, and G6PD deficiency remain visible. Availability of new technologies, such as the biosensor, will fuel ongoing debates about priorities for allocating resources that must be adapted to a constantly evolving target. Technical and logistical concerns regarding the biosensor should be addressed by future product designs, adequate training, strengthened supply chains, and careful planning of communication, advocacy and staff interactions at all health system levels.

Background: Plasmodium spp. asymptomatic carriers are potential reservoirs contributing to the persistence of malaria transmission in endemic areas. The study was designed to assess the extent of Plasmodium spp. asymptomatic infection at household and individual levels and associated risk factors in Northern Côte d’Ivoire.Methods: A cross-sectional survey was conducted in July 2016 at household level in the health district of Korhogo. A questionnaire was administered to household’s head to capture socio-demographic information and practices including malaria treatment and preventive measures. In each household, adults without malaria symptoms nor history of fever during the week before recruitment were screened. Capillary blood samples were collected and used for the detection of Plasmodium spp. infections using both conventional microscopy and a loop-mediated isothermal DNA amplification (LAMP) assay. Logistic regression was used to determine variables that influenced Plasmodium spp. asymptomatic infections.Results: In total, 376 households and 1’011 asymptomatic adults were screened. Asymptomatic Plasmodium spp. infections were identified in 12.5% [47/376] and 38.3% [144/376] of the households and in 5.2% [53/1011] and 18.8% [190/1011] of the individuals screened, according to microscopy and LAMP, respectively. At household level, asymptomatic carriers increased about two times when using mosquito repellent coils compared to those where it is not used (OR: 1.8; p=0.005). At individual level, men’s risk to be infected was about two times that of women (OR: 1.9; pConclusionPlasmodium spp. asymptomatic carriers is important in Northern Côte d’Ivoire and male, age under 30 and perirban living area appear as significant risk factors. Interventions aiming at eliminating asymptomatic infections in this context, should primarily target among other strategy, the at-risk populations and zones.

BackgroundA new more highly sensitive rapid diagnostic test (HS-RDT) forPlasmodium falciparummalaria (Alere™/Abbott Malaria Ag P.f RDT [05FK140], now calledNxTek™Eliminate Malaria Ag Pf) was launched in 2017. The test has already been used in many research studies in a wide range of geographies and use cases.MethodsIn this study, we collate all published and available unpublished studies that use the HS-RDT and assess its performance in (i) prevalence surveys, (ii) clinical diagnosis, (iii) screening pregnant women, and (iv) active case detection. Two individual-level data sets from asymptomatic populations are used to fit logistic regression models to estimate the probability of HS-RDT positivity based on histidine-rich protein 2 (HRP2) concentration and parasite density. The performance of the HS-RDT in prevalence surveys is estimated by calculating the sensitivity and positive proportion in comparison to polymerase chain reaction (PCR) and conventional malaria RDTs.ResultsWe find that across 18 studies, in prevalence surveys, the mean sensitivity of the HS-RDT is estimated to be 56.1% (95% confidence interval [CI] 46.9–65.4%) compared to 44.3% (95% CI 32.6–56.0%) for a conventional RDT (co-RDT) when using nucleic acid amplification techniques as the reference standard. In studies where prevalence was estimated using both the HS-RDT and a co-RDT, we found that prevalence was on average 46% higher using a HS-RDT compared to a co-RDT. For use in clinical diagnosis and screening pregnant women, the HS-RDT was not significantly more sensitive than a co-RDT.ConclusionsOverall, the evidence presented here suggests that the HS-RDT is more sensitive in asymptomatic populations and could provide a marginal improvement in clinical diagnosis and screening pregnant women. Although the HS-RDT has limited temperature stability and shelf-life claims compared to co-RDTs, there is no evidence to suggest, given this test has the same cost as current RDTs, it would have any negative impacts in terms of malaria misdiagnosis if it were widely used in all four population groups explored here.

Background The diagnosis of malaria, using microscopy or rapid diagnostic tests (RDTs), requires the collection of capillary blood. This procedure is relatively simple to perform but invasive and poses potential risks to patients and health workers, arising from the manipulation of potentially infectious bodily fluids. Less or non-invasive diagnostic tests, based on urine, saliva or requiring no sampling, have the potential to generate less discomfort for the patient and to offer simpler and less risky testing procedures that could be safely performed by untrained staff or even self-performed. To explore the potential acceptance and perceived value of such non-invasive tests, an online, international survey was conducted to gather feedback from National Malaria Control Programme (NMCP) representatives. Methods An online survey comprising nineteen questions, available in English, French or Spanish, was emailed to 300 individuals who work with NMCPs in malaria-endemic countries. Answers were collected between November and December 2017; responses were qualitatively analysed to identify key themes and trends and quantitatively analysed to determine average values stratified by region. Results Responses were received from 70 individuals, from 33 countries. Approximately half of the respondents (52 %) considered current blood-based tests for malaria to be minimally invasive and non-problematic in their setting. For these participants, non-invasive tests would only be of interest if they brought additional performance improvements, as compared with the performance of microscopy and RDTs. Most respondents were of the view that saliva-based (80 %) and urine-based (66 %) tests would be more readily acceptable among children than blood-based tests. Potential use-case scenarios of interest for both saliva- and urine-based tests were ease-of-testing by community health workers, additional surveillance, self-testing, and outbreak investigation. Many respondents (41 %) thought that if saliva-based tests retailed at Conclusions Although limited to NMCP stakeholders, this survey indicated that current tests for malaria, based on capillary blood, are generally perceived to be minimally invasive and non-problematic. Non-invasive tests, especially if saliva-based, would be welcome if they could match or out-perform the price and performance of current blood-based tests.

Dried blood spots (DBS) typically prepared on filter papers are an ideal sample type for malaria surveillance by offering easy and cost-effective methods in terms of sample collection, storage, and transport. The objective of this study was to evaluate the applicability of DBS with a commercial multiplex malaria assay, developed to concurrently measure Plasmodium antigens, histidine-rich protein 2 (HRP2), Plasmodium lactate dehydrogenase (pLDH), and a host inflammatory biomarker, C-reactive protein (CRP), in whole blood. The assay conditions were optimized for DBS, and thermal stability for measurement of Plasmodium antigens and CRP in dried blood were determined. Performance of the multiplex assay on matched DBS and whole blood pellet samples was also evaluated using the clinical samples. The results indicate the acceptable performance in multiplex antigen detection using DBS samples. At cutoff levels for DBS, with a diagnostic specificity with a lower 95% confidence bound > 92%, diagnostic sensitivities against polymerase chain reaction (PCR)–confirmed malaria for HRP2, Pf LDH, Pv LDH, and Pan LDH were 93.5%, 80.4%, 21.3%, and 55.6%, respectively. The half-life of pLDH was significantly less than that of HRP2 in thermal stability studies. Results with DBS samples collected from Peru indicate that the uncontrolled storage conditions of DBS can result in inaccurate reporting for infection with P. falciparum parasites with hrp2/3 deletions. With careful consideration that minimizing the unfavorable DBS storage environment is essential for ensuring integrity of heat-labile Plasmodium antigens, DBS samples can be used as an alternative to liquid whole blood to detect P. falciparum with hrp2/3 deletions in malaria surveillance.

Plasmodium vivax is co-endemic with Plasmodium falciparum in Peru, and optimum management requires distinguishing these two species in the blood of patients. For the differential identification of P. vivax and other Plasmodium spp., the LoopampTM Malaria Pan Detection Kit in combination with the Loopamp Malaria Pv Detection Kit (Eiken Chemical Co. Ltd., Tokyo, Japan) was used to evaluate 559 whole blood samples collected in 2017 from febrile patients with suspected malaria attending different health facilities in the Loreto region. The Loopamp Malaria Pan Detection Kit showed a sensitivity of 87.7% (95% CI: 83.5–91.9) and a specificity of 94.4% (95% CI: 91.9–96.9) and good agreement with PCR (Cohen’s kappa 0.8266, 95% CI: 0.7792–0.874). By comparison, the Loopamp Malaria Pv Detection Kit showed a similar sensitivity (84.4%, 95% CI: 79.0–89.7) and specificity (92.4%, 95% CI: 89.7–95.0) and substantial agreement with PCR (Cohen’s kappa: 0.7661, 95% CI: 0.7088–0.8234).

Dried blood spots (DBS) typically prepared on filter papers are an ideal sample type for malaria surveillance by offering easy and cost-effective methods in terms of sample collection, storage, and transport. The objective of this study was to evaluate the applicability of DBS with a commercial multiplex malaria assay, developed to concurrently measure Plasmodium antigens, histidine-rich protein 2 (HRP2), Plasmodium lactate dehydrogenase (pLDH), and a host inflammatory biomarker, C-reactive protein (CRP), in whole blood. The assay conditions were optimized for DBS, and thermal stability for measurement of Plasmodium antigens and CRP in dried blood were determined. Performance of the multiplex assay on matched DBS and whole blood pellet samples was also evaluated using the clinical samples. The results indicate the acceptable performance in multiplex antigen detection using DBS samples. At cutoff levels for DBS, with a diagnostic specificity with a lower 95% confidence bound > 92%, diagnostic sensitivities against polymerase chain reaction (PCR)–confirmed malaria for HRP2, Pf LDH, Pv LDH, and Pan LDH were 93.5%, 80.4%, 21.3%, and 55.6%, respectively. The half-life of pLDH was significantly less than that of HRP2 in thermal stability studies. Results with DBS samples collected from Peru indicate that the uncontrolled storage conditions of DBS can result in inaccurate reporting for infection with P. falciparum parasites with hrp2/3 deletions. With careful consideration that minimizing the unfavorable DBS storage environment is essential for ensuring integrity of heat-labile Plasmodium antigens, DBS samples can be used as an alternative to liquid whole blood to detect P. falciparum with hrp2/3 deletions in malaria surveillance. Supplementary Information The online version of this article (10.1007/s12639-020-01325-2) contains supplementary material, which is available to authorized users.

International audience; A major gap in the Plasmodium vivax elimination toolkit is the identification of individuals carrying clinically silent and undetectable liver-stage parasites, called hypnozoites. This study developed a panel of serological exposure markers capable of classifying individuals with recent P. vivax infections who have a high likelihood of harboring hypnozoites. We measured IgG antibody responses to 342 P. vivax proteins in longitudinal clinical cohorts conducted in Thailand and Brazil and identified candidate serological markers of exposure. Candidate markers were validated using samples from year-long observational cohorts conducted in Thailand, Brazil and the Solomon Islands and antibody responses to eight P. vivax proteins classified P. vivax infections in the previous 9 months with 80% sensitivity and specificity. Mathematical models demonstrate that a serological testing and treatment strategy could reduce P. vivax prevalence by 59-69%. These eight antibody responses can serve as a biomarker, identifying individuals who should be targeted with anti-hypnozoite therapy.

Background The production and use of malaria rapid diagnostic tests (RDTs) has risen dramatically over the past 20 years. In view of weak or non-existing in vitro diagnostics (IVD) regulations and post-marketing surveillance (PMS) systems in malaria endemic countries, the World Health Organization, later joined by the Foundation for Innovative New Diagnostics, established an independent, centralized performance evaluation and Lot Testing (LT) programme to safeguard against poor quality of RDTs being distributed through the public health sector of malaria endemic countries. RDT performances and manufacturer quality management systems have evolved over the past decade raising questions about the future need for a centralized LT programme. Results Between 2007 and 2017, 6056 lots have been evaluated, representing approximately 1.6 Billion RDTs. A total of 69 lots (1.1%) failed the quality control. Of these failures, 26 were detected at receipt of the RDT lot in the LT laboratory, representing an estimated 7.9 million poor quality RDTs, and LT requesters were advised that RDTs were not of sufficient quality for use in patient management. Forty-three were detected after long-term storage in the laboratory, of which 24 (56%) were found to be due to a major issue with insufficient buffer volume in single use buffer vials, others predominantly showing loss of sensitivity. The annual cost of running the programme, based on expenses recorded in years 2014–2016, an estimated volume of 700 lots per year and including replenishment of quality control samples, was estimated at US$ 178,500 ($US 255 per lot tested). Conclusions Despite the clear benefits of the centralized LT programme and its low cost compared with the potential costs of each country establishing its own PMS system for RDTs, funding concerns have made its future beyond 2020 uncertain. In order to manage the risks of misdiagnosis due to low quality RDTs, and to ensure the continued safety and reliability of malaria case management, there is a need to ensure that an effective and implementable approach to RDT quality control continues to be available to programmes in endemic countries.

Background Malaria diagnostics by rapid diagnostic test (RDT) relies primarily on the qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and Plasmodium spp lactate dehydrogenase (pLDH). As novel RDTs with increased sensitivity are being developed and implemented as point of care diagnostics, highly sensitive laboratory-based assays are needed for evaluating RDT performance. Here, a quantitative suspension array technology (qSAT) was developed, validated and applied for the simultaneous detection of PfHRP2 and pLDH in a variety of biological samples (whole blood, plasma and dried blood spots) from individuals living in different endemic countries. Results The qSAT was specific for the target antigens, with analytical ranges of 6.8 to 762.8 pg/ml for PfHRP2 and 78.1 to 17076.6 pg/ml for P. falciparum LDH (Pf-LDH). The assay detected Plasmodium vivax LDH (Pv-LDH) at a lower sensitivity than Pf-LDH (analytical range of 1093.20 to 187288.5 pg/ml). Both PfHRP2 and pLDH levels determined using the qSAT showed to positively correlate with parasite densities determined by quantitative PCR (Spearman r = 0.59 and 0.75, respectively) as well as microscopy (Spearman r = 0.40 and 0.75, respectively), suggesting the assay to be a good predictor of parasite density. Conclusion This immunoassay can be used as a reference test for the detection and quantification of PfHRP2 and pLDH, and could serve for external validation of RDT performance, to determine antigen persistence after parasite clearance, as well as a complementary tool to assess malaria burden in endemic settings.

Background A novel ultrasensitive malaria rapid diagnostic test (us-RDT) has been developed for improved active Plasmodium falciparum infection detection. The usefulness of this us-RDT in clinical diagnosis and fever management has not been evaluated. Methods Diagnostic performance of us-RDT was compared retrospectively to that of conventional RDT (co-RDT) in 3000 children and 515 adults presenting with fever to Tanzanian outpatient clinics. The parasite density was measured by an ultrasensitive qPCR (us-qPCR), and the HRP2 concentration was measured by an enzyme-linked immunosorbent assay. Results us-RDT identified few additional P. falciparum–positive patients as compared to co-RDT (276 vs 265 parasite-positive patients detected), with only a marginally greater sensitivity (75% vs 73%), using us-qPCR as the gold standard (357 parasite-positive patients detected). The specificity of both RDTs was >99%. Five of 11 additional patients testing positive by us-RDT had negative results by us-qPCR. The HRP2 concentration was above the limit of detection for co-RDT (>3653 pg of HRP2 per mL of blood) in almost all infections (99% [236 of 239]) with a parasite density >100 parasites per µL of blood. At parasite densities 793 pg/mL) and co-RDT in 29 (25%) and 24 (20%) of 118 patients, respectively. Conclusion There is neither an advantage nor a risk of using us-RDT, rather than co-RDT, for clinical malaria diagnosis. In febrile patients, only a small proportion of infections are characterized by a parasite density or an HRP2 concentration in the range where use of us-RDT would confer a meaningful advantage over co-RDT., Ultrasensitive rapid diagnostic tests (RDTs) for malaria, designed for Plasmodium falciparum elimination campaigns, may be used off label in clinical practice. The diagnostic performances of ultrasensitive and conventional RDTs were similar in febrile Tanzanian outpatients, which is explained by the underlying HRP2 concentration and parasite density distributions.

In order to accelerate towards malaria elimination, improved targeting of limited resources is essential. A major gap in our elimination toolkit forPlasmodium vivaxmalaria is the identification of individuals carrying arrested liver stages, called hypnozoites. These clinically silent but frequently relapsing hypnozoites are key toP. vivaxpersistence. Whilst hypnozoites cannot be directly detected, individuals who have had recent exposure toP. vivaxand have not been treated are likely to harbor these parasites. By measuring IgG antibody responses to over 300P. vivaxproteins, a panel of serological markers capable of detecting exposure toP. vivaxinfections in the prior 9-month period was identified and validated. Using antibody responses to 8P. vivaxproteins, 80% sensitivity and specificity for detecting recent infections were achieved in three independent studies conducted in Thailand, Brazil and the Solomon Islands. As these individuals have a high likelihood of harboring hypnozoites, the suite of these 8 antibody responses can serve as biomarkers for the identification of individuals who should be targeted for treatment with liver-stage drugs such as primaquine and tafenoquine in mass drug administration programs aimed at controlling and eliminatingP. vivaxmalaria.One Sentence SummaryThe manuscript describes identification and validation of a novel panel of P. vivax proteins that can be used to detect recent exposure to P. vivax infections within the prior 9 months.

Background Pregnant women frequently show low-density Plasmodium infections that require more sensitive methods for accurate diagnosis and early treatment of malaria. This is particularly relevant in low-malaria transmission areas, where intermittent preventive treatment is not recommended. Molecular methods, such as polymerase chain reaction (PCR) are highly sensitive, but require sophisticated equipment and advanced training. Instead, loop mediated isothermal amplification (LAMP) provides an opportunity for molecular detection of malaria infections in remote endemic areas, outside a reference laboratory. The aim of the study is to evaluate the performance of LAMP for the screening of malaria in pregnant women in Colombia. Methods This is a nested prospective study that uses data and samples from a larger cross-sectional project conducted from May 2016 to January 2017 in three Colombian endemic areas (El Bagre, Quibdó, and Tumaco). A total of 531 peripheral and placental samples from pregnant women self-presenting at local hospitals for antenatal care visits, at delivery or seeking medical care for suspected malaria were collected. Samples were analysed for Plasmodium parasites by light microscopy (LM), rapid diagnostic test (RDT) and LAMP. Diagnostic accuracy endpoints (sensitivity, specificity, predictive values, and kappa scores) of LM, RDT and LAMP were compared with nested PCR (nPCR) as the reference standard. Results In peripheral samples, LAMP showed an improved sensitivity (100.0%) when compared with LM 79.5% and RDT 76.9% (p

International audience; Antimalarial drug resistance is a major constraint for malaria control and elimination efforts. Artemisinin-based combination therapy is now the mainstay for malaria treatment. However, delayed parasite clearance following treatment with artemisinin derivatives has now spread in the Greater Mekong Sub region and may emerge or spread to other malaria endemic regions. This spread is of great concern for malaria control programmes, as no alternatives to artemisinin-based combination therapies are expected to be available in the near future. There is a need to strengthen surveillance systems for early detection and response to the antimalarial drug resistance threat. Current surveillance is mainly done through therapeutic efficacy studies; however these studies are complex and both time- and resource-intensive. For multiple common antimalarials, parasite drug resistance has been correlated with specific genetic mutations, and the molecular markers associated with antimalarial drug resistance offer a simple and powerful tool to monitor the emergence and spread of resistant parasites. Different techniques to analyse molecular markers associated with antimalarial drug resistance are available, each with advantages and disadvantages. However, procedures are not adequately harmonized to facilitate comparisons between sites. Here we describe the target product profiles for tests to analyse molecular markers associated with antimalarial drug resistance, discuss how use of current techniques can be standardised, and identify the requirements for an ideal product that would allow malaria endemic countries to provide useful spatial and temporal information on the spread of resistance.

Background Accurate and efficient detection of sub-microscopic malaria infections is crucial for enabling rapid treatment and interruption of transmission. Commercially available malaria LAMP kits have excellent diagnostic performance, though throughput is limited by the need to prepare samples individually. Here, we evaluate the clinical performance of a newly developed high throughput (HTP) sample processing system for use in conjunction with the Eiken malaria LAMP kit. Methods The HTP system utilised dried blood spots (DBS) and liquid whole blood (WB), with parallel sample processing of 94 samples per run. The system was evaluated using 699 samples of known infection status pre-determined by gold standard nested PCR. Results The sensitivity and specificity of WB-HTP-LAMP was 98.6% (95% CI, 95.7–100), and 99.7% (95% CI, 99.2–100); sensitivity of DBS-HTP-LAMP was 97.1% (95% CI, 93.1–100), and specificity 100% against PCR. At parasite densities greater or equal to 2 parasites/μL, WB and DBS HTP-LAMP showed 100% sensitivity and specificity against PCR. At densities less than 2 p/μL, WB-HTP-LAMP sensitivity was 88.9% (95% CI, 77.1–100) and specificity was 99.7% (95% CI, 99.2–100); sensitivity and specificity of DBS-HTP-LAMP was 77.8% (95% CI, 54.3–99.5) and 100% respectively. Conclusions The HTP-LAMP system is a highly sensitive diagnostic test, with the potential to allow large scale population screening in malaria elimination campaigns.

BACKGROUND: Rapid diagnostic tests (RDTs) are today the most widely used method for malaria diagnosis and are recommended, alongside microscopy, for the confirmation of suspected cases before the administration of anti-malarial treatment. The diagnostic performance of RDTs, as compared to microscopy or PCR is well described but the actual analytical sensitivity of current best-in-class tests is poorly documented. This value is however a key performance indicator and a benchmark value needed to developed new RDTs of improved sensitivity. METHODS: Thirteen RDTs detecting either the Plasmodium falciparum histidine rich protein 2 (HRP2) or the plasmodial lactate dehydrogenase (pLDH) antigens were selected from the best performing RDTs according to the WHO-FIND product testing programme. The analytical sensitivity of these products was evaluated using a range of reference materials including P. falciparum and Plasmodium vivax whole parasite samples as well as recombinant proteins. RESULTS: The best performing HRP2-based RDTs could detect all P. falciparum cultured samples at concentrations as low as 0.8 ng/mL of HRP2. The limit of detection of the best performing pLDH-based RDT specifically detecting P. vivax was 25 ng/mL of pLDH. CONCLUSION: The analytical sensitivity of P. vivax and Pan pLDH-based RDTs appears to vary considerably from product to product, and improvement of the limit-of-detection for P. vivax detecting RDTs is needed to match the performance of HRP2 and Pf pLDH-based RDTs for P. falciparum. Different assays using different reference materials produce different values for antigen concentration in a given specimen, highlighting the need to establish universal reference assays.

The global prevalence of malaria has decreased over the past fifteen years, but similar gains have not been realized against Plasmodium vivax because this species is less responsive to conventional malaria control interventions aimed principally at P. falciparum. Approximately half of all malaria cases outside of Africa are caused by P. vivax. This species places dormant forms in human liver that cause repeated clinical attacks without involving another mosquito bite. The diagnosis of acute patent P. vivax malaria relies primarily on light microscopy. Specific rapid diagnostic tests exist but typically perform relatively poorly compared to those for P. falciparum. Better diagnostic tests are needed for P. vivax. To guide their development, FIND, in collaboration with P. vivax experts, identified the specific diagnostic needs associated with this species and defined a series of three distinct target product profiles, each aimed at a particular diagnostic application: (i) point-of-care of acutely ill patients for clinical care purposes; (ii) point-of-care asymptomatic and otherwise sub-patent residents for public health purposes, e.g., mass screen and treat campaigns; and (iii) ultra-sensitive not point-of-care diagnosis for epidemiological research/surveillance purposes. This report presents and discusses the rationale for these P. vivax-specific diagnostic target product profiles. These contribute to the rational development of fit-for-purpose diagnostic tests suitable for the clinical management, control and elimination of P. vivax malaria., Author summary Plasmodium vivax is the second most prevalent Plasmodium species amongst the five that can infect humans and cause malaria. The control and elimination of P. vivax is complicated by its specific biology, such as hard-to-detect low densities of blood-circulating parasites in infected individuals, the existence of persistent liver forms causing relapse, or the early appearance of sexual stages of the parasite during the course of an infection, which facilitates its transmission. These difficulties are reinforced by the fact that most antimalarial tools have been developed primarily for P. falciparum, the most prevalent malaria species, and are not always as effective for P. vivax. Current tools for the diagnosis of P. vivax are of limited effectiveness. Rapid diagnostic tests exist but show, in average, lower performance than similar test for P. falciparum. P. vivax diagnosis often relies on light microscopy which is challenging to maintain at a high quality and not sensitive enough to detect a large fraction of all infections. Recognizing that better diagnostic tools for P. vivax are needed, we report in this study the development of new target product profiles to define the specific characteristics of such tests. The establishment of these consensus-based documents is an important first step to guide research and development efforts toward better diagnostic solutions for P. vivax malaria and to accelerate the elimination of this species alongside P. falciparum.

Regulated by histone acetyltransferases and deacetylases (HDACs), histone acetylation is a key epigenetic mechanism controlling chromatin structure, DNA accessibility, and gene expression. HDAC inhibitors induce growth arrest, differentiation, and apoptosis of tumor cells and are used as anticancer agents. Here we describe the effects of HDAC inhibitors on microbial sensing by macrophages and dendritic cells in vitro and host defenses against infection in vivo. HDAC inhibitors down-regulated the expression of numerous host defense genes, including pattern recognition receptors, kinases, transcription regulators, cytokines, chemokines, growth factors, and costimulatory molecules as assessed by genome-wide microarray analyses or innate immune responses of macrophages and dendritic cells stimulated with Toll-like receptor agonists. HDAC inhibitors induced the expression of Mi-2β and enhanced the DNA-binding activity of the Mi-2/NuRD complex that acts as a transcriptional repressor of macrophage cytokine production. In vivo, HDAC inhibitors increased the susceptibility to bacterial and fungal infections but conferred protection against toxic and septic shock. Thus, these data identify an essential role for HDAC inhibitors in the regulation of the expression of innate immune genes and host defenses against microbial pathogens.

Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and luciferase assays, that validation by targeted proteomics substantially enriched for biologically relevant let-7 interactors. For example, we found that the zinc finger protein ZTF-7 was a bona fide let-7 miRNA target. We also validated predicted miR-58 targets, demonstrating that this approach is adaptable to other miRNAs. We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational methods or in large-scale experiments, and that the described strategy should be readily adaptable to other organisms.

Background Pregnancy poses specific challenges for the diagnosis of Plasmodium falciparum infection due to parasite sequestration in the placenta, which translates in low circulation levels in peripheral blood. The aim of this study is to assess the performance of a new highly sensitive rapid diagnostic test (HS-RDT) for the detection of malaria in peripheral and placental blood samples from pregnant women in Colombia. Methods This is a retrospective study using 737 peripheral and placental specimens collected from pregnant women in Colombian malaria-endemic regions. Light microscopy (LM), conventional rapid diagnostic tests (Pf/Pv RDT and Pf RDT), and HS-RDT testing were performed. Diagnostic accuracy endpoints of LM, HS-RDT and RDTs were compared with nested polymerase chain reaction (nPCR) as the reference test. Results In comparison with nPCR, the sensitivity of HS-RDT, Pf RDT, Pf/Pv RDT and LM to detect infection in peripheral samples was 85.7% (95% CI = 70.6–93.7), 82.8% (95% CI = 67.3–91.9), 77.1% (95% CI = 61.0–87.9) and 77.1% (95% CI = 61.0–87.9) respectively. The sensitivity to detect malaria in asymptomatic women, was higher with HS-RDT, where LM and Pf/Pv RDT missed half of infections detected by nPCR, but differences were not significant. Overall, specificity was similar for all tests (>99.0%). In placental blood, the prevalence of infection by P. falciparum by nPCR was 2.8% (8/286), by HS-RDT was 1% and by conventional RDTs (Pf RDT and Pf/Pv RDT) and LM was 0.7%. The HS-RDT detected placental infections in peripheral blood that were negative by LM and Pf/Pv RDT, however the number of positive placentas was low. Conclusions The sensitivity of HS-RDT to detect P. falciparum infections in peripheral and placental samples from pregnant women was slightly better compared to routinely used tests during ANC visits and at delivery. Although further studies are needed to guide recommendations on the use of the HS-RDT for malaria case management in pregnancy, this study shows the potential value of this test to diagnose malaria in pregnancy in low-transmission settings.

MicroRNAs (miRNAs) repress target genes through a poorly defined antisense mechanism. Cell-free and cell-based assays have supported the idea that miRNAs repress their target mRNAs by blocking initiation of translation, whereas studies in animal models argued against this possibility. We examined endogenous targets of the let-7 miRNA, an important regulator of stem cell fates. We report that let-7 represses translation initiation in Caenorhabditis elegans, demonstrating this mode of action for the first time in an organism. Unexpectedly, although the lin-4 miRNA was previously reported to repress its targets at a step downstream of translation initiation, we also observe repression of translation initiation for this miRNA. This repressive mechanism, which frequently but not always coincides with transcript degradation, requires the GW182 proteins AIN-1 and AIN-2, and acts on several mRNAs targeted by different miRNAs. Our analysis of an expanded set of endogenous miRNA targets therefore indicates widespread repression of translation initiation under physiological conditions and establishes C. elegans as a genetic system for dissection of the underlying mechanisms.

Contains fulltext : 80470.pdf (Publisher’s version ) (Closed access) The cytokine macrophage migration inhibitory factor plays a central role in inflammation, cell proliferation and tumorigenesis. Moreover, macrophage migration inhibitory factor levels correlate with tumor aggressiveness and metastatic potential. Histone deacetylase inhibitors are potent antitumor agents recently introduced in the clinic. Therefore, we hypothesized that macrophage migration inhibitory factor would represent a target of histone deacetylase inhibitors. Confirming our hypothesis, we report that histone deacetylase inhibitors of various chemical classes strongly inhibited macrophage migration inhibitory factor expression in a broad range of cell lines, in primary cells and in vivo. Nuclear run on, transient transfection with macrophage migration inhibitory factor promoter reporter constructs and transduction with macrophage migration inhibitory factor expressing adenovirus demonstrated that trichostatin A (a prototypical histone deacetylase inhibitor) inhibited endogenous, but not episomal, MIF gene transcription. Interestingly, trichostatin A induced a local and specific deacetylation of macrophage migration inhibitory factor promoter-associated H3 and H4 histones which did not affect chromatin accessibility but was associated with an impaired recruitment of RNA polymerase II and Sp1 and CREB transcription factors required for basal MIF gene transcription. Altogether, this study describes a new molecular mechanism by which histone deacetylase inhibitors inhibit MIF gene expression, and suggests that macrophage migration inhibitory factor inhibition by histone deacetylase inhibitors may contribute to the antitumorigenic effects of histone deacetylase inhibitors.

MicroRNAs (miRNAs) are small noncoding RNAs that control diverse cellular and developmental events through repression of large sets of target mRNAs. Regulated transcription of the genes encoding miRNAs by RNA polymerase II promotes specific expression patterns of individual miRNAs. However, recent studies have established that substantial regulation of mature miRNA accumulation also occurs after transcription. Here, we review the mechanisms of such post-transcriptional regulation, with a particular focus on examples where molecular mechanisms or physiological principles are beginning to emerge. Elucidating these mechanisms will increase our understanding of gene regulation and provide new insights into causes of miRNA misexpression in diseases such as cancer.

MicroRNAs (miRNAs) are noncoding RNAs that regulate numerous target genes through a posttranscriptional mechanism and thus control major developmental pathways. The phylogenetically conserved let-7 miRNA regulates cell proliferation and differentiation, thus functioning as a key regulator of developmental timing in C. elegans and a tumor suppressor gene in humans. Using a reverse genetic screen, we have identified genetic interaction partners of C. elegans let-7, including known and novel potential target genes. Initial identification of several translation initiation factors as suppressors of a let-7 mutation led us to systematically examine genetic interaction between let-7 and the translational machinery, which we found to be widespread. In the presence of wild-type let-7, depletion of the translation initiation factor eIF3 resulted in precocious cell differentiation, suggesting that developmental timing is translationally regulated, possibly by let-7. As overexpression of eIF3 in humans promotes translation of mRNAs that are also targets of let-7-mediated repression, we suggest that eIF3 may directly or indirectly oppose let-7 activity. This might provide an explanation for the opposite functions of let-7 and eIF3 in regulating tumorigenesis.

Malaria is one of the most significant causes of childhood mortality, but disease control efforts are threatened by resistance of the Plasmodium parasite to current therapies. Continued progress in combating malaria requires development of new, easy to administer drug combinations with broad-ranging activity against all manifestations of the disease. DSM265, a triazolopyrimidine-based inhibitor of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH), is the first DHODH inhibitor to reach clinical development for treatment of malaria. We describe studies profiling the biological activity, pharmacological and pharmacokinetic properties, and safety of DSM265, which supported its advancement to human trials. DSM265 is highly selective toward DHODH of the malaria parasite Plasmodium, efficacious against both blood and liver stages of P. falciparum, and active against drug-resistant parasite isolates. Favorable pharmacokinetic properties of DSM265 are predicted to provide therapeutic concentrations for more than 8 days after a single oral dose in the range of 200 to 400 mg. DSM265 was well tolerated in repeat-dose and cardiovascular safety studies in mice and dogs, was not mutagenic, and was inactive against panels of human enzymes/receptors. The excellent safety profile, blood- and liver-stage activity, and predicted long half-life in humans position DSM265 as a new potential drug combination partner for either single-dose treatment or once-weekly chemoprevention. DSM265 has advantages over current treatment options that are dosed daily or are inactive against the parasite liver stage.

Plasmodium falciparum , the most deadly agent of malaria, displays a wide variety of resistance mechanisms in the field. The ability of antimalarial compounds in development to overcome these must therefore be carefully evaluated to ensure uncompromised activity against real-life parasites. We report here on the selection and phenotypic as well as genotypic characterization of a panel of sensitive and multidrug-resistant P. falciparum strains that can be used to optimally identify and deconvolute the cross-resistance signals from an extended panel of investigational antimalarials. As a case study, the effectiveness of the selected panel of strains was demonstrated using the 1,2,4-oxadiazole series, a newly identified antimalarial series of compounds with in vitro activity against P. falciparum at nanomolar concentrations. This series of compounds was to be found inactive against several multidrug-resistant strains, and the deconvolution of this signal implicated pfcrt , the genetic determinant of chloroquine resistance. Targeted mode-of-action studies further suggested that this new chemical series might act as falcipain 2 inhibitors, substantiating the suggestion that these compounds have a site of action similar to that of chloroquine but a distinct mode of action. New antimalarials must overcome existing resistance and, ideally, prevent its de novo appearance. The panel of strains reported here, which includes recently collected as well as standard laboratory-adapted field isolates, is able to efficiently detect and precisely characterize cross-resistance and, as such, can contribute to the faster development of new, effective antimalarial drugs.

Background Loop-mediated isothermal DNA amplification (LAMP) methodology offers an opportunity for point-of-care (POC) molecular detection of asymptomatic malaria infections. However, there is still little evidence on the feasibility of implementing this technique for population screenings in isolated field settings. Methods Overall, we recruited 1167 individuals from terrestrial (‘road’) and hydric (‘riverine’) communities of the Peruvian Amazon for a cross-sectional survey to detect asymptomatic malaria infections. The technical performance of LAMP was evaluated in a subgroup of 503 samples, using real-time Polymerase Chain Reaction (qPCR) as reference standard. The operational feasibility of introducing LAMP testing in the mobile screening campaigns was assessed based on field-suitability parameters, along with a pilot POC-LAMP assay in a riverine community without laboratory infrastructure. Results LAMP had a sensitivity of 91.8% (87.7–94.9) and specificity of 91.9% (87.8–95.0), and the overall accuracy was significantly better among samples collected during road screenings than riverine communities (p≤0.004). LAMP-based diagnostic strategy was successfully implemented within the field-team logistics and the POC-LAMP pilot in the riverine community allowed for a reduction in the turnaround time for case management, from 12–24 hours to less than 5 hours. Specimens with haemolytic appearance were regularly observed in riverine screenings and could help explaining the hindered performance/interpretation of the LAMP reaction in these communities. Conclusions LAMP-based molecular malaria diagnosis can be deployed outside of reference laboratories, providing similar performance as qPCR. However, scale-up in remote field settings such as riverine communities needs to consider a number of logistical challenges (e.g. environmental conditions, labour-intensiveness in large population screenings) that can influence its optimal implementation.

Background New field applicable diagnostic tools are needed for highly sensitive detection of residual malaria infections in pre-elimination settings. Field performance of a high throughput DNA extraction system for loop mediated isothermal amplification (HTP-LAMP) was therefore evaluated for detecting malaria parasites among asymptomatic individuals in Zanzibar. Methods HTP-LAMP performance was evaluated against real-time PCR on 3008 paired blood samples collected on filter papers in a community-based survey in 2015. Results The PCR and HTP-LAMP determined malaria prevalences were 1.6% (95% CI 1.3-2.4) and 0.7% (95% CI 0.4-1.1), respectively. The sensitivity of HTP-LAMP compared to PCR was 40.8% (CI95% 27.0-55.8) and the specificity was 99.9% (CI95% 99.8-100). For the PCR positive samples, there was no statistically significant difference between the geometric mean parasite densities among the HTP-LAMP positive (2.5 p/mu L, range 0.2-770) and HTP-LAMP negative (1.4 p/mu L, range 0.1-7) samples (p = 0.088). Two lab technicians analysed up to 282 samples per day and the HTP-LAMP method was experienced as user friendly. Conclusions Although field applicable, this high throughput format of LAMP as used here was not sensitive enough to be recommended for detection of asymptomatic low-density infections in areas like Zanzibar, approaching malaria elimination.

Novel synthetic endoperoxides are being evaluated as new components of artemisinin combination therapies (ACTs) to treat artemisinin-resistant Plasmodium falciparum malaria. We conducted blinded ex vivo activity testing of fully synthetic (OZ78 and OZ277) and semisynthetic (artemisone, artemiside, artesunate, and dihydroartemisinin) endoperoxides in the histidine-rich protein 2 enzyme-linked immunosorbent assay against 200 P. falciparum isolates from areas of artemisinin-resistant malaria in western and northern Cambodia in 2009 and 2010. The order of potency and geometric mean (GM) 50% inhibitory concentrations (IC 50 s) were as follows: artemisone (2.40 nM) > artesunate (8.49 nM) > dihydroartemisinin (11.26 nM) > artemiside (15.28 nM) > OZ277 (31.25 nM) > OZ78 (755.27 nM). Ex vivo activities of test endoperoxides positively correlated with dihydroartemisinin and artesunate. The isolates were over 2-fold less susceptible to dihydroartemisinin than the artemisinin-sensitive P. falciparum W2 clone and showed sensitivity comparable to those with test endoperoxides and artesunate, with isolate/W2 IC 50 susceptibility ratios of P. falciparum chloroquine resistance transporter mutations, with negative correlations in sensitivity to endoperoxides and chloroquine. The activities of endoperoxides (artesunate, dihydroartemisinin, OZ277, and artemisone) significantly correlated with that of the ACT partner drug, mefloquine. Isolates had mutations associated with clinical resistance to mefloquine, with 35% prevalence of P. falciparum multidrug resistance gene 1 ( pfmdr1 ) amplification and 84.5% occurrence of the pfmdr1 Y184F mutation. GM IC 50 s for mefloquine, lumefantrine, and endoperoxides (artesunate, dihydroartemisinin, OZ277, OZ78, and artemisone) correlated with pfmdr1 copy number. Given that current ACTs are failing potentially from reduced sensitivity to artemisinins and partner drugs, newly identified mutations associated with artemisinin resistance reported in the literature and pfmdr1 mutations should be examined for their combined contributions to emerging ACT resistance.

Malaria is still considered a deadly scourge in Africa, Asia, and South America despite improved vector control and curative treatments with new antimalarial combinations. The next challenge is to work towards disease eradication. To achieve this goal it is crucial to develop, validate, and integrate biological assays into test cascades that align with the key target product profiles. For anti-relapse, a parent molecule should kill hypnozoites or cause activation of Plasmodium vivax liver stages. For transmission blocking, dual equal-activity antimalarials killing both the asexual and the sexual parasite stages in human blood are favored. Finally, by assessing cross resistance and generating drug resistance in the laboratory, it is expected that new medicines with acceptable resistance profiles will be forthcoming.

BACKGROUND: Recent whole cell in vitro screening campaigns identified thousands of compounds that are active against asexual blood stages of Plasmodium falciparum at submicromolar concentrations. These hits have been made available to the public, providing many novel chemical starting points for anti-malarial drug discovery programmes. Knowing which of these hits are fast-acting compounds is of great interest. Firstly, a fast action will ensure rapid relief of symptoms for the patient. Secondly, by rapidly reducing the parasitaemia, this could minimize the occurrence of mutations leading to new drug resistance mechanisms.An in vitro assay that provides information about the speed of action of test compounds has been developed by researchers at GlaxoSmithKline (GSK) in Spain. This assay also provides an in vitro measure for the ratio between parasitaemia at the onset of drug treatment and after one intra-erythrocytic cycle (parasite reduction ratio, PRR). Both parameters are needed to determine in vitro killing rates of anti-malarial compounds. A drawback of the killing rate assay is that it takes a month to obtain first results. METHODS: The approach described in the present study is focused only on the speed of action of anti-malarials. This has the advantage that initial results can be achieved within 4-7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly. It is expected that this new assay can be used as a filter in the early drug discovery phase, which will reduce the number of compounds progressing to secondary, more time-consuming assays like the killing rate assay. RESULTS: The speed of action of a selection of seven anti-malarial compounds was measured with two independent experimental procedures using modifications of the standard [3H]hypoxanthine incorporation assay. Depending on the outcome of both assays, the tested compounds were classified as either fast or non-fast-acting. CONCLUSION: The results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds. Another advantage of the approach is its ability to discriminate between static or cidal compound effects.