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

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

Abstract Background Malaria rapid diagnostic tests (RDTs) are largely responsible for the gains made in the proportion of malaria cases confirmed with a parasitological test. However, quality assurance programs to support their use remain a challenge. A dried tube specimen (DTS) method was developed that showed potential for use as a stable source of quality control (QC) sample for RDTs and for use in external quality assessments or proficiency testing (PT). DTS was further assessed with focus on sample stability under field settings in Benin and Liberia. Methods DTS were prepared using Plasmodium falciparum 3D7 or W2 strains at concentrations of 1000, 500 or 0 parasites/µL and tested for baseline reactivity at the Centers for Disease Control and Prevention, Atlanta before shipping. In Benin and Liberia, DTS were stored under refrigeration in a reference laboratory (RL) or in health centres under ambient temperatures. Seven rounds of testing were performed at 4-week intervals during which DTS were tested on RDTs stored at the RL or at health centres. Observed DTS reactivity at the RL and health centres were compared to expected reactivity to determine DTS stability. DTS were also assembled into a PT panel and tested by health facility staff at the mid and end time-points of the study. Daily maximum and minimum storage temperatures for RDTs and DTS were recorded. Results In Benin, DTS, irrespective of storage conditions, produced the expected reactivity at all time points. However, evidence of degradation was observed at weeks 20 and 24 for DTS stored at ambient temperatures at the health centres and not those stored under refrigeration at the RL. In Liberia, sample degradation was observed starting at week 8 especially among DTS stored at the health facilities. The degradation was associated with prolonged storage of DTS under ambient temperature prior to study commencement and less than optimal storage temperatures at the RL. Use of DTS in a PT enabled identification of health worker errors in performing the tests. Conclusion DTS is a feasible tool for use as QC material and for PT under field conditions. Long-term (> 5 months) storage of DTS requires refrigeration.

Background: Malaria rapid diagnostic tests (RDTs) are largely responsible for the gains made in the proportion of malaria cases confirmed with a parasitological test. However, quality assurance programs to support their use remain a challenge. A dried tube specimen (DTS) method was developed that showed potential for use as a stable source of quality control (QC) sample for RDTs and for use in external quality assessments or proficiency testing (PT). DTS was further assessed with focus on sample stability under field settings in Benin and Liberia. Methods: DTS were prepared using Plasmodium falciparum 3D7 or W2 strains at concentrations of 1000, 500 or 0 parasites/µL and tested for baseline reactivity at the Centers for Disease Control and Prevention, Atlanta before shipping. In Benin and Liberia, DTS were stored under refrigeration in a reference laboratory (RL) or in health centres under ambient temperatures. Seven rounds of testing were performed at 4-week intervals during which DTS were tested on RDTs stored at the RL or at health centres. Observed DTS reactivity at the RL and health centres were compared to expected reactivity to determine DTS stability. DTS were also assembled into a PT panel and tested by health facility staff at the mid and end time-points of the study. Daily maximum and minimum storage temperatures for RDTs and DTS were recorded. Results: In Benin, DTS, irrespective of storage conditions, produced the expected reactivity at all time points. However, evidence of degradation was observed at weeks 20 and 24 for DTS stored at ambient temperatures at the health centres and not those stored under refrigeration at the RL. In Liberia, sample degradation was observed starting at week 8 especially among DTS stored at the health facilities. The degradation was associated with prolonged storage of DTS under ambient temperature prior to study commencement and less than optimal storage temperatures at the RL. Use of DTS in a PT enabled identification of health worker errors in performing the tests. Conclusion: DTS is a feasible tool for use as QC material and for PT under field conditions. Long-term (> 5 months) storage of DTS requires refrigeration. [ABSTRACT FROM AUTHOR]

Abstract Background In malaria elimination settings, the very low levels of transmission now being attained present challenges that demand new strategies to identify and treat low-density infections in both symptomatic and asymptomatic populations. Accordingly, passive case detection activities need to be supplemented by active case detection (ACD) strategies with more sensitive diagnostic tools. Malaria rapid diagnostic tests (RDTs) have provided low- and middle-income countries with unprecedented access to malaria diagnostics. Nevertheless, conventional RDTs miss a potentially important proportion of sub-microscopic infections. Therefore, new combination highly sensitive (HS-)RDTs, able to detect low parasite densities and identify all infected individuals, could support countries implementing ACD strategies for radical cure to accelerate malaria elimination. To address this need, an on-line survey was conducted to gather information from malaria control programme representatives to guide the development of next-generation RDTs. Results Most of respondents confirmed that ACD was a common activity in their programmes (56/75; 75%). Although microscopy was the preferred method in case management and reactive case detection, RDTs were the primary diagnostic tests used in proactive case detection (31/75; 41%). In terms of preferences for species detection in a new combination HS-RDT, data was not one-directional. Survey respondents slightly preferred the Pf/Pv/Pan combination (42%; 21/50), while Pf/Pan was more popular among end-users. Survey respondents also valued a low-cost (

Background: In malaria elimination settings, the very low levels of transmission now being attained present challenges that demand new strategies to identify and treat low-density infections in both symptomatic and asymptomatic populations. Accordingly, passive case detection activities need to be supplemented by active case detection (ACD) strategies with more sensitive diagnostic tools. Malaria rapid diagnostic tests (RDTs) have provided low- and middle-income countries with unprecedented access to malaria diagnostics. Nevertheless, conventional RDTs miss a potentially important proportion of sub-microscopic infections. Therefore, new combination highly sensitive (HS-) RDTs, able to detect low parasite densities and identify all infected individuals, could support countries implementing ACD strategies for radical cure to accelerate malaria elimination. To address this need, an on-line survey was conducted to gather information from malaria control programme representatives to guide the development of nextgeneration RDTs. Results: Most of respondents confirmed that ACD was a common activity in their programmes (56/75; 75%). Although microscopy was the preferred method in case management and reactive case detection, RDTs were the primary diagnostic tests used in proactive case detection (31/75; 41%). In terms of preferences for species detection in a new combination HS-RDT, data was not one-directional. Survey respondents slightly preferred the Pf/Pv/Pan combination (42%; 21/50), while Pf/Pan was more popular among end-users. Survey respondents also valued a low-cost (< $1.00 USD), lightweight and portable test, able to detect asymptomatic infections and differentiate species, as well as provide immediate results that could be interpreted with the naked eye. In addition, respondents were open to new tests and even to replace the existing ones for ACD (63%; 47/75). Conclusions: This survey provided valuable information on the use and current limitations of ACD, on the primary product characteristics for a next-generation combination HS-RDT to support ACD and radical cure, and on the potential adoption of such a test, if available, to support malaria elimination. [ABSTRACT FROM AUTHOR]

Background: Plasma Plasmodium falciparum histidine-rich protein-2 (PfHRP2) is the most accurate biomarker for severe malaria, but its measurement by ELISA has been considered too unwieldy to incorporate into clinical management. Methods: Plasma samples covering a wide range of PfHRP2 concentrations were applied to rapid diagnostic tests (RDTs). RDTs were read by eye and digital capture, and PfHRP2 concentrations were measured via serial dilution with results compared to ELISA readings. Results: The Paracheck® brand showed the strongest correlation (r2 = 0.963) as well as the lowest inter-observer variability (combined kappa across band intensities for three observers = 0.938). Plasma PfHRP2 measurement via serial dilution showed minimal bias compared to ELISA and acceptable limits of agreement. Three different dilutions of a well characterized set of admission samples from uncomplicated and severe malaria patients studied in a low transmission setting gave an area under the receiver operator characteristic curve of 0.844 in terms of identifying severe malaria. Conclusions: These studies show that plasma PfHRP2 can be assessed via a single RDT, with application of a plasma dilution of 1:5 or 1:10 providing useful diagnostic information to assist in patient management or clinical trial inclusion. [ABSTRACT FROM AUTHOR]

Background: Rapid diagnostic tests (RDTs) are now widely used for laboratory confirmation of suspected malaria cases to comply with the World Health Organization recommendation for universal testing before treatment. However, many malaria programmes lack quality control (QC) processes to assess RDT use under field conditions. Prior research showed the feasibility of using the dried tube specimen (DTS) method for preserving Plasmodium falciparum parasites for use as QC samples for RDTs. This study focused on the use of DTS for RDT QC and proficiency testing under field conditions. Methods: DTS were prepared using cultured P. falciparum at densities of 500 and 1,000 parasites/|L; 50 |L aliquots of these along with parasite negative human blood controls (0 parasites/|L) were air-dried in specimen tubes and reactivity verified after rehydration. The DTS were used in a field study in the Oromia Region of Ethiopia. Replicate DTS samples containing 0, 500 and 1,000 parasites/|L were stored at 4°C at a reference laboratory and at ambient temperatures at two nearby health facilities. At weeks 0, 4, 8, 12, 16, 20, and 24, the DTS were rehydrated and tested on RDTs stored under manufacturer-recommended temperatures at the RL and on RDTs stored under site-specific conditions at the two health facilities. Reactivity of DTS stored at 4°C at the reference laboratory on RDTs stored at the reference laboratory was considered the gold standard for assessing DTS stability. A proficiency-testing panel consisting of one negative and three positive samples, monitored with a checklist was administered at weeks 12 and 24. Results: At all the seven time points, DTS stored at both the reference laboratory and health facility were reactive on RDTs stored under the recommended temperature and under field conditions, and the DTS without malaria parasites were negative. At the reference laboratory and one health facility, a 500 parasites/|L DTS from the proficiency panel was falsely reported as negative at week 24 due to errors in interpreting faint test lines. Conclusions: The DTS method can be used under field conditions to supplement other RDT QC methods and health worker proficiency in Ethiopia and possibly other malaria-endemic countries. [ABSTRACT FROM AUTHOR]

Background: Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing. Methods: A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay. Results: The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined. Conclusions: Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in resource-limited settings. The system requires none or only little instrumentation for the nucleic acid amplification reaction and the read-out is possible with the naked eye. Showing the same sensitivity and specificity as comparable diagnostic methods but simultaneously increasing reaction speed and dramatically reducing assay requirements, the method has potential to become a true point-of-care test for the malaria parasite. [ABSTRACT FROM AUTHOR]

The article focuses on the introduction of mobile phones into the rural Bandarban district of Bangladesh to improve case detection and treatment of patients with malaria. It offers information on the implementation of a malarial control program by the Government of Bangladesh and Bangladesh Rural Advancement Committee (BRAC) for early diagnosis of Plasmodium falciparum malaria using rapid diagnostic tests (RDT) and artemisinin combination therapy (ACT).

Background: Rapid diagnostic tests (RDT) for malaria have been demonstrated to be effective and they should replace microscopy in certain areas. Method: The cost-effectiveness of five RDT and thick smear microscopy was estimated and compared. Data were collected on Brazilian Extra-Amazon Region. Data sources included the National Malaria Control Programme of the Ministry of Health, the National Healthcare System reimbursement table, laboratory suppliers and scientific literature. The perspective was that of the Brazilian public health system, the analytical horizon was from the start of fever until the diagnostic results provided to patient and the temporal reference was that of year 2010. Two costing methods were produced, based on exclusive-use microscopy or shared-use microscopy. The results were expressed in costs per adequately diagnosed cases in 2010 U.S. dollars. One-way sensitivity analysis was performed considering key model parameters. Results: In the cost-effectiveness analysis with exclusive-use microscopy, the RDT CareStartTM was the most cost-effective diagnostic strategy. Microscopy was the most expensive and most effective, with an additional case adequately diagnosed by microscopy costing US$ 35,550.00 in relation to CareStartTM. In opposite, in the cost-effectiveness analysis with shared-use microscopy, the thick smear was extremely cost-effective. Introducing into the analytic model with shared-use microscopy a probability for individual access to the diagnosis, assuming a probability of 100% of access for a public health system user to any RDT and, hypothetically, of 85% of access to microscopy, this test saw its effectiveness reduced and was dominated by the RDT CareStartTM. Conclusion: The analysis of cost-effectiveness of malaria diagnosis technologies in the Brazilian Extra-Amazon Region depends on the exclusive or shared use of the microscopy. Following the assumptions of this study, shared-use microscopy would be the most cost-effective strategy of the six technologies evaluated. However, if used exclusively for diagnosing malaria, microscopy would be the worst use of resources. Microscopy would not be the most cost-effective strategy, even when structure is shared with other programmes, when the probability of a patient having access to it was reduced. Under these circumstances, the RDT CareStartTM would be the most cost-effective strategy. [ABSTRACT FROM AUTHOR]