Date Published: January 25, 2011
Publisher: Public Library of Science
Abstract: The Malaria Eradication Research Agenda (malERA) Consultative Group on Diagnoses and Diagnostics outline a research and development agenda to provide the diagnosis and diagnostic tools required for malaria eradication.
Partial Text: As malaria transmission declines across much of its range and the possibility of elimination (reduction of transmission to zero in a defined geographical area) is increasingly considered ,, accurate diagnosis and case identification through the demonstration of malaria parasites in sick patients presenting to health workers (“passive case detection”) is ever more important. During case management in all settings, all symptomatic patients with demonstrated parasitemia should be considered to be malaria cases, and all parasitemic patients should be given definitive antimalarial treatment. Accurate diagnosis is essential both to target antimalarial drugs and to enable effective management of the frequently fatal nonmalarial febrile illnesses  that share signs and symptoms with malaria –.
Identification of parasitemia in febrile patients is essential in all of the programmatic phases of the continuum from malaria control to elimination, although the challenges for health systems in maintaining this activity in areas where malaria has become rare will be more prominent, as will the importance of detecting asymptomatic infections of low parasite density. The ongoing role of other routine interventions, such as intermittent preventive treatment in pregnancy, needs reevaluating as elimination is approached. Moreover, because the distribution of malaria transmission is often highly heterogeneous within a country, strategies may need to vary at a subnational level. Analyses of past experiences and operations research are required to guide decisions on when these changes in emphasis should take place as control progresses ,. Although programs in areas of higher transmission will be less likely to engage in active case finding of individuals with low parasite densities, surveillance is nevertheless necessary to detect trends and the impact of interventions, and requires appropriate, high-throughput diagnostic tools. In addition to the diagnosis of malaria, it will be critical to have diagnostic capabilities for other causes of presenting illness, particularly fever. A sick adult or parent of a febrile child may not be satisfied with a diagnosis of “not malaria,” and both patients and providers require guidance on the integrated management of childhood illnesses, to ensure that appropriate alternative and specific treatment is available and provided.
Once malaria is eliminated in a given area, considerable resources will be required to detect reintroduction through surveillance and to maintain capacity for rapid management and investigation of any cases found, as long as the risk factors that support transmission are still in place. Screening of migrant populations, screening of populations around detected cases, and case management tools for screening suspected patients, such as recent travelers or geographical associates of malaria cases may be needed. The tools to achieve these activities must be readily available in an environment where technicians are likely to be unskilled in the use of malaria diagnostic tests, particularly microscopy . Thus, the requirements for surveillance and screening in areas where malaria has been eliminated, but risk of transmission is present, are similar to those of programs in an elimination phase. However, case management tools that are minimally dependent on previous technician experience in diagnosing malaria will be of particular importance.
In settings where there is risk of autochthonous or imported malaria, diagnostics must be capable of rapidly and accurately detecting and quantifying parasitemia in febrile patients, and identifying species. In addition, highly sensitive diagnostic tools are needed for passive case detection and case management at health care facilities (public or private) that report to the national health information or disease surveillance systems. The issues around diagnostics in both case management and surveillance and control settings have a large impact on, and are impacted by, monitoring and evaluation requirements and health systems implementation issues such as the development of improved supply lines and logistics management, reporting of results and commodity consumption, and adherence of health workers and patients to management consistent with diagnostic results. These are all important areas where pooling of knowledge and sometimes operational research is required to maximize the impact of the diagnostic tools discussed below ,.
When performed to a high standard, light microscopy is capable of accurately identifying and quantifying Plasmodium parasites with sufficient rapidity for case management in most settings. It remains the operational gold standard in both control and elimination settings. However, the quality of light microscopy in the field is often inadequate – and limited by factors such as the instability and difficult preparation of currently used Romanowsky-based stains –, poorly maintained, low quality equipment, and inadequate training, supervision, and quality assurance. Additionally, as malaria transmission decreases, it is likely that light microscopy technician skills may be redeployed elsewhere. Consequently, research into sustainable ways to maintain high-quality light microscopy in field settings, including innovative training, supervisory, and quality-assurance systems, is badly needed. More consistent and stable staining techniques are also required. This area of research has been ignored for the past 60 to 100 years, but has the potential to improve field accuracy significantly and may also improve the potential of the new reading techniques discussed below. Large volumes of slides pose particular challenges with respect to reading, especially in settings with low parasite prevalence where microscopist performance is hard to maintain .
Computer-assisted analysis of Giemsa-stained slides (possibly combined with automated staining), or digitized image transfer (potentially via mobile telephone) to a reference centre for review by an expert microscopist may enable greater consistency in parasite detection –. Additional research is required to determine whether these techniques will detect lower parasite densities than can be obtained by traditional light microscopy. Related techniques under development use software analysis of the scatter of various wavelengths of light to identify Plasmodium parasites and other pathogens. Although these digital techniques have the potential to improve field detection of malaria parasites, field-ready versions are not yet available, and it is not known whether these tools will meet the requirements for use in resource-poor settings.
Fluorescent-assisted microscopy (FAM)-based methods—for example, the quantitative buffy coat (QBC) method , incorporation of a fluorescent probe (fluorescence in situ hybridization [FISH]) or of parasite DNA , or antigen staining—has been used to a limited extent in various programs. FAM methods may eventually speed up slide reading and reduce operator error. High-throughput FAM may become possible if high specificity can be maintained by the absence of low artifactual staining. However, at present FAM cannot differentiate between species, a capability considered a major advantage of light microscopy over today’s antigen-detection tests, although species-specific markers for FISH assays and fluorescent-tagged monoclonal antibodies are being developed. In addition, the applicability of FAM to parasite quantitation is not clear and FAM requires specialized equipment that will limit where it can be used.
RDTs based on the detection of specific parasite antigens that use a platform design of lateral immunochromatographic flow (dipsticks or plastic cassettes) have started to change the way malaria is diagnosed in endemic settings. RDTs are increasingly being used at the community level and in control programs for case management and in prevalence surveys. Good RDTs reliably detect parasitemia down to 100–200 parasites/µl, which is comparable to the sensitivity of routine well-performed light microscopy . In general, RDTs are simple to use. With training and quality assurance, they can be used by peripheral facility and village health workers to determine whether malaria parasites are present in a patient. However, increasing use in field settings suggests that many commercial RDTs have variable detection thresholds and field stability . Systems for monitoring performance and routine quality control of manufactured product lots are therefore required.
Standardized quality-control methods for RDTs are important for confirming test quality and ensuring that health workers and patients trust results. As with microscopy , quality assurance of RDTs requires a comprehensive, organized program ,. Such programs are absent in many countries. The development of standardized panels containing known concentrations of target antigens will greatly broaden the reach, applicability, and sustainability of RDT quality-control programs. Parasite-based panels that use cryo-preserved parasite preparations  are currently available at a centralized (regional) level, but panels that are easier to standardize and widely available are needed. Likewise, standardized regulatory approval and procurement in keeping with best practices will reduce the requirement for investment by individual procurement agencies in quality control and product evaluation programs. The development of low-cost tools for confirming quality at the national and field level (positive controls ) is also necessary to improve reach and sustainability. Finally, novel approaches that use PCR to confirm RDT results might eventually be useful.
For use in active surveillance and case finding, a diagnostic tool must be suitable for use in resource-poor field settings. Diagnostic tests must therefore be supportable at the district level or below, be affordable and low-maintenance, require less operator training than current methods, and have a low requirement for consumables. They should also detect very low parasite densities and distinguish between all locally prevalent Plasmodium species, be minimally invasive, and provide sufficiently rapid results to facilitate effective case management when an infection is identified. For use in prevalence surveys, where immediate management of asymptomatic parasitemia is not the aim, testing at a more centralized level may be sufficient. But, even in this context, rapid feedback and case management are desirable.
Current methods of detecting circulating parasites by demonstrating parasite DNA through amplification of ribosomal RNA (rRNA) genes by PCR assays represent the overall gold standard of malaria diagnostics. When sample concentration methods are used, 0.5 parasite/µl unconcentrated blood or lower can be detected. Quantitative PCR can be used to determine the concentration of circulating DNA and therefore estimate the density of circulating parasites. Survey and testing techniques, including pooling of samples, can reduce costs  but also reduce sensitivity to some extent by diluting samples.
Hemozoin, a by-product of Plasmodium metabolism, can be detected through refraction/absorbance of laser light of certain frequencies, and has been used to detect malaria and to determine species. Current field-ready technologies are based on flow cytometers. Their application is limited to screening, however, because of low sensitivity at low parasite densities –. Current research activities include the development of transcutaneous hemozoin detection. If sufficiently sensitive and specific, this approach might offer a noninvasive test for malaria for mass-population screening of, for example, individuals moving into a malaria elimination area. Hemozoin detection may find a place in routine case management if appropriate tools can be developed.
Current antigen-detecting RDTs (see earlier for details) are likely to miss a significant proportion of asymptomatic cases in low-transmission settings ,,,. Thus, although the current generation of RDTs can indicate the presence of malaria in a community, they cannot determine the true prevalence of parasite carriage. Research aimed towards increasing the sensitivity of existing RDTs may not change this situation because of the limitations of the currently available technology. Some antigen-detecting ELISAs are more sensitive than RDTs. Furthermore, because they can also be used to quantify antigen, they have been used to monitor drug efficacy. Antigen-detecting ELISAs may also facilitate high-throughput testing. However, their use is currently limited by laboratory and training requirements.
Antibody detection (see also ) is currently available in ELISA and RDT formats, and is a sensitive way to demonstrate past exposure to malaria parasites (past infection). Because antibodies may not be detectable in blood-stage infections of very recent onset, these tests are inappropriate for case management. However, they may be useful in detecting established P. falciparum infections in which the blood-stage parasite density has fallen below the limits of light microscopy or antigen-detecting RDTs . Detection of antisporozoite antibodies (so-called anti-CSP antibodies) alone or in combination with antibodies to blood-stage parasites has also been suggested as a surrogate for detecting individuals with a high likelihood of carrying P. vivax hypnozoites (evidence of infection) –. However, anti-CSP antibody responses are usually low and transient, especially in areas of low and moderate transmission, which renders this test unreliable.
The central importance of active case detection in each programmatic stage towards elimination has been comprehensively dealt with by several of the other malERA Consultative Groups –. However, whether active case detection can be achieved at sufficiently high and sustainable levels will depend to a great extent on the field utility and costs of the diagnostic and other tools eventually adopted for this role and on how these tests are used.
Malaria elimination in the most challenging settings will require improvements in point-of-care tests for case management, and the development of new tests capable of identifying very low parasite densities in asymptomatic individuals in field settings for mass screening and treatment. As a result of our discussions, we propose a research and development agenda for diagnoses and diagnostics that should stimulate and facilitate the development, validation, and use of such tests (see Box 1).