5

Diagnostic Tests

WHERE WE WANT TO BE IN THE YEAR 2010

There will be new appreciation of the need for different malaria diagnostics at various levels of the health care system. Malaria assays will be available—each tailored to a distinct setting—that will supplement microscopy for diagnosing malaria. Microscopic examination of blood films, when used, will be simpler and more accurate as a result of improved equipment and protocols and because microscopists will be better trained. Most of the new malaria diagnostic tests will be inexpensive and simple to use, and they will provide results in less than an hour. Each will be sensitive to low parasitemias and will be able to distinguish among different parasite species. Nearly all of the assays will depend on minimally invasive specimen collection techniques (a disposable lancet for finger-prick blood sampling will be universally used in the field, greatly decreasing concerns about transmitting AIDS and hepatitis B) and will be able to be batched for high-volume diagnostic situations. Mass screening of blood slides, which often results in large backlogs of unread slides, will give way to focused and well-planned surveys of malaria prevalence. Finally, tests will soon be marketed that distinguish between the presence of parasites and the presence of disease in semi-immune individuals, and other tests will be developed that will identify those patients at risk of progressing to severe illness.



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MALARIA: Obstacles and Opportunities 5 Diagnostic Tests WHERE WE WANT TO BE IN THE YEAR 2010 There will be new appreciation of the need for different malaria diagnostics at various levels of the health care system. Malaria assays will be available—each tailored to a distinct setting—that will supplement microscopy for diagnosing malaria. Microscopic examination of blood films, when used, will be simpler and more accurate as a result of improved equipment and protocols and because microscopists will be better trained. Most of the new malaria diagnostic tests will be inexpensive and simple to use, and they will provide results in less than an hour. Each will be sensitive to low parasitemias and will be able to distinguish among different parasite species. Nearly all of the assays will depend on minimally invasive specimen collection techniques (a disposable lancet for finger-prick blood sampling will be universally used in the field, greatly decreasing concerns about transmitting AIDS and hepatitis B) and will be able to be batched for high-volume diagnostic situations. Mass screening of blood slides, which often results in large backlogs of unread slides, will give way to focused and well-planned surveys of malaria prevalence. Finally, tests will soon be marketed that distinguish between the presence of parasites and the presence of disease in semi-immune individuals, and other tests will be developed that will identify those patients at risk of progressing to severe illness.

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MALARIA: Obstacles and Opportunities WHERE WE ARE TODAY The most important role for malaria diagnostics is to help health care workers, whether they be in a village in Mali or in a sophisticated hospital in New York City, select the most appropriate treatment for patients whose illness may be due to infection with malaria parasites. Since the symptoms of malaria vary and can resemble those of other diseases, diagnosing malaria solely on the basis of clinical symptoms is unreliable. If evidence of malaria is found, particularly in nonimmune individuals, rapid and appropriate therapy is essential to prevent further progression of the disease. Alternatively, if parasites are absent, other explanations for the symptoms must be sought so that appropriate treatment can be started and the use of toxic antimalarial drugs can be avoided. The simple microscopic diagnostic tests available today have two drawbacks, even when performed correctly. In patients who live in malarious areas and who are partially immune to the disease, malaria infections may be asymptomatic and of little clinical significance. The presence of parasites in such patients cannot be assumed to cause the symptoms of an illness, which may have other causes. There is currently no diagnostic to associate malaria infection with disease in such patients. In addition, repeated blood films may be necessary to detect malaria parasites in nonimmune patients, in whom symptoms can arise from very low parasitemias. Failure to detect parasites in a single blood film from such a patient (for instance, an American recently returned from a malarious area) cannot be used to exclude a diagnosis of malaria. Diagnostic tests are also important for certain types of epidemiologic surveys. Used for this purpose, rapidity and pinpoint accuracy are not as critical as the need for the safe collection, preparation, and evaluation of a large number of samples. Because of the danger of transmitting other diseases, such as AIDS or hepatitis B, through the use of contaminated lancets, the collection of blood samples raises significant biosafety issues. In addition, the transportation and examination of blood slides for epidemiologic surveys can be both cumbersome and logistically difficult, particularly in remote areas. In many countries, there is a backlog of slides to be examined, and results may not be available for many months. The relevance of out-of-date results to the planning and evaluation of malaria field operations is questionable. Currently, the “gold standard” for diagnosing malaria in individual patients and for epidemiologic surveys is the microscopic examination of blood smears. The presence of malaria parasites, identified by their characteristic morphology, is considered definitive proof of infection. A number of potential improvements to current microscopic methods and alternatives to microscopy for diagnosing malaria are discussed below.

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MALARIA: Obstacles and Opportunities Microscopy Standard Technique Microscopy is the most widely used laboratory-based diagnostic test for malaria, and it likely will remain the test of choice for some time. In this technique, two drops of blood, typically obtained from a finger pricked by a metal lancet, are placed on a glass microscope slide. One drop is smeared to create a thin blood film, the other drop (the thick film) is left alone, and the slide is allowed to air-dry. The cells in the thin blood smear are chemically fixed to the slide, and the slide is stained with Giemsa or some other stain formulation to facilitate detection of parasites. The water in the staining solution lyses the unfixed red blood cells in the thick blood film, removing the hemoglobin, and white blood cells and any malaria parasites are fixed to the slide. The thick blood film allows the microscopist to look for parasites in a relatively large volume of blood, thus increasing the sensitivity of the test. The thin blood film, which better preserves parasite morphology, is used to quantify and identify parasites to the species level, if necessary. A high-power microscope (400 times to 1,000 times magnification, with an oil immersion objective) is required to read thick and thin blood films. Between 100 and 200 microscope fields must be examined to rule out the presence of parasites in a thick blood film. The ability to detect infection by microscopy depends on the number of fields inspected and the experience of the technician reading the slide. An experienced microscopist can evaluate and make a diagnosis from a slide from a heavily parasitized individual within about a minute; additional time is required to detect parasites on a blood film from a lightly parasitized individual (Payne, 1988). Microscopy has proved to be a tremendously resilient and useful diagnostic tool, but it is not without problems. The cost of materials (slides and staining reagents) is relatively low, but substantial investment is required to purchase microscopes and to select and train technicians; additional funds are needed for maintenance of the microscopes. Microscopy is labor-intensive, and a high level of technical skill is required for correct preparation and interpretation of slides. This means that the workload of each technician must be monitored, since fatigue or the pressure to return results can lead to a significant loss of efficiency and accuracy. In addition, many microscopes in the field, through age, deterioration, and hard use, are nearing or are at the end of their useful working lives. High-quality microscopes are expensive and often beyond the means of regional health outposts. Few if any peripheral health facilities have access to sturdy and portable microscopes required for field use.

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MALARIA: Obstacles and Opportunities Quantitative Buffy Coat Technique The quantitative buffy coat (QBC) technique is a commercially available test based on fluorescence microscopy (Wardlaw and Levine, 1983). The test uses a specially made glass capillary tube of precise internal diameter containing acridine orange as a vital stain. After the tube is filled with blood, it is capped and a small plastic float is inserted. The float displaces precisely 90 percent of the interior tube space along its length, and when centrifuged, settles at the plasma-red blood cell interface, physically expanding the length of the buffy coat layer 10-fold. White blood cell components appear as discrete bands and can be accurately quantified with a specially designed optical device. The float also extends into and expands the top portion of the red blood cell layer where the parasitized red blood cells, because of their lower density, are concentrated. The centrifuged tube is observed directly, using a fluorescence microscope. Since the contrast between stain and background is high, parasitized red blood cells are easily seen. QBC Versus Microscopy QBC is a relatively new technique, and the limits of its sensitivity and specificity are still being explored. In a study in Ethiopia, microscopists detected 10 percent more infections by QBC than by conventional slide methods (Spielman et al., 1988). By contrast, a similar study of QBC in Thailand found the technique to be only marginally more sensitive than conventional microscopy and of acceptable specificity (Tharavanij, 1990). Another study found QBC to be as sensitive as microscopy in experimentally infected volunteers, highly sensitive when used to diagnose malaria infection in hospital patients, but less sensitive than microscopy when used for mass screening (Rickman et al., 1989). QBC is a quick and efficient method of processing batches of blood specimens. Its advantage in this regard is less apparent, however, when a microscopist who is an expert at interpreting blood smears for malaria infection is available. In resource-limited settings, the cost of the capillary tubes and the need for additional equipment, such as a centrifuge and fluorescence microscope, make QBC less attractive. Most facilities in malaria-endemic countries do not have access to fluorescence microscopes. Alternatives to Microscopy There is a recognized need for dependable alternatives to diagnostic microscopy for detecting the presence of malaria infection. Tests that are as simple and accurate as microscopy but that require less sophisticated

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MALARIA: Obstacles and Opportunities equipment and training would be particularly valuable in field settings. In addition, the medical community's lack of experience in the use of microscopy to diagnose malaria in the United States (and other more developed countries) makes alternative tests potentially valuable for use in domestic hospitals. Research efforts to identify malaria parasite antigens and genes, which could provide the basis for such tests, have spurred the development of a number of new diagnostic methods, through the use of monoclonal antibodies and recombinant DNA techniques, which have been reviewed in detail elsewhere (Bruce-Chwatt, 1987; World Health Organization, 1988; Pammenter, 1988; Wirth et al., 1989; Tharavanij, 1990). These assays are designed to detect specific parasite antigens or nucleic acids with limits of detection equal to or better than that provided by microscopy. The development of many initially promising assays often has languished because of the difficulty of reproducing the results of laboratory research in field trials. Development has also been slowed by the challenge of creating a simple assay and because improved diagnostic tests have not been a priority for public funding in malaria research. Immunoassays Immunological techniques that detect malaria parasite antigens have been described since the early 1980s. These tests would be useful in health care facilities where microscopy is not performed, and they could supplement or replace microscopy in other settings. Antigen detection techniques would also be useful epidemiologic tools, providing data for community-based public health intervention measures. Ideal target antigens for immunological assays should not persist in the blood (or in other specimens, such as urine that might be tested) after the parasite disappears, should be abundant in blood or other clinical specimens to maximize test sensitivity, and should be genus and species specific, without cross-reactivity with host antigens or antigens from other microorganisms (World Health Organization, 1988). Experimental tests for detecting malaria parasite antigens are based on both antigen-competition and antigen-capture formats, using both enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) methods. Researchers have described the use of both monoclonal antibodies and polyclonal antisera in assays for genus-specific antigens, such as those that cross-react with Plasmodium berghei (Avidor et al., 1985), undefined P. falciparum antigens (Mackey et al., 1982; Avraham et al., 1983; Khusmith et al., 1987), and defined malarial antigens (Fortier et al., 1987), and in idiotype-anti-idiotype detection systems (Zhou and Li, 1987). Studies of RIA- and ELISA-based tests have demonstrated detection of very low densities of parasitized red blood cells (in the range of 0.01 to

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MALARIA: Obstacles and Opportunities 0.001 percent, equivalent to 50 to 500 parasites per microliter of blood), with RIA techniques being slightly more sensitive on average. In general, therefore, the limits of detection of immunoassay techniques approach that of microscopy (which is able to discern 10 to 20 parasites per microliter of blood). The value of RIA methods for both immunoassays and probes (see Genetic-Probe Assays, below) has in the past been to verify that laboratory tests can be developed by using certain combinations of reagents and malarial antigens. However, because RIAs employ radioisotopes that have short shelf lives and are costly and potentially dangerous, they are impractical for routine clinical use, even at the central-hospital level in the developing world. In addition, laboratory personnel have considerably more experience and training in the application of ELISA methods, especially since the start of the AIDS epidemic. Although diagnostic ELISA kits have been used successfully at many well-equipped central health care facilities and in support of epidemiologic or vaccine programs, they are not suited for use in outlying areas where equipment is lacking. The minimum of two to four hours needed to perform an ELISA, and the desirability of testing large numbers of specimens at one time, means that same-day results may not always be available. This is not, however, a problem in large epidemiologic studies, in which same-day results are rarely required. Antigen Inhibition and Competition In antigen-inhibition and antigen-competition assays, parasite antigens are coated on a surface, such as a microwell plate. The test specimen is centrifuged, and the red blood cells are washed and lysed to extract parasite antigen. The specimen is then mixed with a high-titer serum or monoclonal antibody and placed in the well containing test antigen. If the specimen contains parasite antigens, they will bind to the antimalaria antibodies, thus preventing the antibodies from binding to the antigens on the plate. After rinsing, the amount of antibody bound to the plate is determined by standard RIA or ELISA techniques (Khusmith et al., 1987). Antigen Capture In antigen-capture (antibody-sandwich) assays, polyclonal or monoclonal antibody is adsorbed to a tube or microwell plate. The specimen is placed into the test vessel and, after washing, a second antibody source conjugated with an enzyme or radioactive label is added. If malarial antigens are present, they serve as a bridge between the antibodies in the tube or well of the plate and the labeled antibodies. The plate is then processed by RIA or ELISA as described above, and the amount of labeling is quantified. Antigen-capture assays may prove to be more practical than antigen-competition assays for diagnosing malaria. Unlike competition assays, capture methods do not require purified malarial antigens and have a greater potential for detecting a broader range of antigens. In

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MALARIA: Obstacles and Opportunities addition, competition assays usually require centrifugation to separate red blood cells from autologous serum antibodies that can interfere with the assay and produce false-positive results. This requirement for specimen processing makes performing large numbers of competition assays time-consuming and limits the technique's usefulness in the field. Soluble Antigens Researchers have identified soluble, parasite-specific antigens in sera from persons infected with malarial parasites (McGregor et al., 1968; Bein and Olcen, 1984; Taylor et al., 1986). Assays have been developed that target some of these antigens (Fortier et al., 1987; Parra and Taylor, 1989). The advantage of assays that detect soluble antigens is that plasma from uncoagulated whole blood can be used without extracting the red blood cells or pretreating the specimen. This type of assay system should be able to diagnosis malaria even in the absence of circulating infected red blood cells, a situation sometimes found in cerebral malaria cases. Genetic-Probe Assays Genetic-probe assays utilize DNA hybridization to detect parasite-specific nucleic acid sequences in red blood cell specimen extracts. Typically, red blood cells are lysed to extract nucleic acids either before or after being fixed on a filter membrane. The membrane is heated to denature the nucleic acids, blocked with nonspecific DNA, and incubated with a specific radiolabeled or biotinylated probe. Although biotinylated probes have been used most often, other modified bases are available. The membranes are then washed and developed, either by autoradiography or by enzyme-substrate labeling. Ideally, these assays could serve as single-use diagnostics and be capable of being batched for higher volume epidemiologic or public health applications. Whole P. falciparum DNA derived from laboratory-cultured organisms has been radiolabeled in vitro to produce a species-specific test. The entire genome was used as a probe, and the resulting assay had a sensitivity equivalent to that of microscopy (Pollack et al., 1985). Although sensitive, such tests have a low specificity, do not allow identification of parasites to the species level, and may cross-react with human DNA. The time needed to complete the assay (usually 24 to 48 hours) and the requirement to prepare large quantities of radiolabeled probes from cultured organisms are additional drawbacks. Some researchers have used cloned DNA fragments as genetic probes (Franzen et al., 1984; Zolg et al., 1987). Others have used synthetic oligonucleotides from genomic regions containing conserved, 21-base-pair tandem repeat sequences (Barker et al., 1986; Holmberg et al., 1986; McLaughlin et al., 1987a,b; Sethabutr et al., 1988). The repeat

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MALARIA: Obstacles and Opportunities sequences which they target are found in abundance in parasite DNA (104 to 105 copies per nucleus). Tests of the 21-oligonucleotide repeat probe in clinical trials in Thailand and Kenya revealed a sensitivity of between 82 and 89 percent and a specificity of between 99 and 94 percent, respectively, compared with microscopy (Barker et al., 1989a,b). Others, using a similar oligonucleotide probe, claim 100 percent specificity and sensitivity of 68 percent (Holmberg et al., 1987). With low levels of parasitemia, however, current DNA probes are less sensitive than microscopy (Lanar et al., 1989). Probes using ribosomal RNA (rRNA) have been used to identify various microorganisms, including plasmodia, since each parasite species has distinct small subunit rRNA nucleotide sequences (Lal et al., 1989; Waters and McCutchan, 1989). Because rRNA constitutes between 85 and 95 percent of the total parasite complement of nucleic acid, synthetic rRNA oligonucleotide probes promise to be very sensitive. Data on rRNA probes specific for all four human Plasmodium species have been reported, but they have not been validated on clinical samples (Waters and McCutchan, 1989). For all nucleic acid-based probes, radiolabeling has produced the greatest sensitivity. However, radioisotope-based assays are impractical for field use because of cost, short shelf life, hazards, and the complex equipment required. To overcome this problem, colorimetric probes are being developed and field tested (McLaughlin et al., 1987a). These probes bind enzyme-avidin conjugates and use ELISA-type enzyme-substrate systems to generate signals. Unfortunately, the hybridization procedure is complex, making it cumbersome in the field. Even the best enzyme-probe systems require equipment not often available outside of central health facilities, such as a slot-blot apparatus, vacuum pump, constant-temperature water bath or incubator, and oven, in addition to standard laboratory glassware. Polymerase Chain Reaction Technologies that can amplify specific sequences of DNA offer great potential when applied to the diagnosis of malaria. The polymerase chain reaction (PCR) method, for example, amplifies specific DNA segments by repeated cycles of heat denaturation, annealing with defined DNA primer pairs, and primer extension mediated by heat-resistant DNA polymerase isolated from a thermophilic bacterium. Polymerase chain reaction techniques require 25 to 30 rounds of amplification, which take three to four hours. Electrical power is needed to run a thermal cycler, and the reagents are relatively expensive. There is a recent report of an ELISA-type assay for PCR products, making the detection of PCR results at least theoreti-

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MALARIA: Obstacles and Opportunities cally possible in less developed settings (Nickerson et al., 1990). The technique offers a hypothetical sensitivity limit of a single malaria gene per specimen, but in practice it requires 10 to 100 gene copies. Problems with contamination from parasite DNA that was not originally in the sample and difficulties in specimen processing need to be resolved before PCR can be used routinely as a diagnostic tool. In addition, personnel who perform the test must be highly trained, and the costs of PCR thermal cyclers and materials remain high. Improvements in PCR technology, such as rapid sample preparation, packaging of reagents to prevent cross-contamination, and the development of nonradioactive signal systems, are bringing the technique closer to the stage where it will be of practical use. The PCR technique has been used to diagnose malaria infection with DNA primers specific for P. falciparum and P. vivax (W. J. Martin, University of Southern California Medical Center, unpublished data, 1991). By sequencing the dihydrofolate reductase gene associated with pyrimethamine resistance, specific primer sequences have been identified that react with the mutant gene but not the wild-type gene. These sequences potentially could be used for clinical diagnosis, identification of the parasite to the species level, and determination of pyrimethamine resistance. Unless results can be accurately quantified and correlated with parasitemia, PCR may not be the test of choice in areas where malaria is endemic and where the majority of inhabitants harbor low levels of parasites without disease. The optimal use of PCR may be in sophisticated clinical laboratories in developed countries that have expertise in using complex technology but are unable to maintain competency in malaria microscopy. PCR may also be useful as an epidemiologic tool. For example, the technique could be used to determine the extent of falciparum malaria in an area of mixed infection and to identify drug-resistant strains in patient specimens or in mosquitoes. Antibody Assays Serological assays can detect antimalarial antibodies but cannot determine whether the antibodies result from current or past infection. Therefore, such assays are not appropriate for diagnosis but can be used for certain epidemiologic applications (Voller and Draper, 1982). Several assay methods, including indirect immunofluorescence, hemagglutination, ELISA, and avidin-biotin-peroxidase complex ELISA, have been tested for their ability to diagnose malaria. Assays have been developed to measure antibodies to crude parasite antigens (Demedts et al., 1987; Sato et al., 1990); antigamete antibodies, which may influence parasite infectivity (Mendis et al., 1987); antibodies to merozoite surface anti-

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MALARIA: Obstacles and Opportunities gen 1, which may indicate acute infection (Früh et al., 1989); and antibodies to circumsporozoite proteins as a broad screen for malaria prevalence (Wirtz et al., 1987). Comparison of the sensitivities and specificities of these assays has been difficult since the various protocols and antigen extracts have not been standardized. To overcome the difficulties associated with producing a standardized, contaminant-free supply of antigen, recombinant DNA techniques have been used to identify, clone, and express or synthesize epitopes of parasite proteins. Among these are the asparagine-alanine-asparagine-proline (NANP) repeat sequences of the P. falciparum circumsporozoite protein (Del Giudice et al., 1987; Knobloch et al., 1987). Synthetic peptide antigen generated by these amino acid repeat sequences was used to develop an ELISA method that identified 80 percent of the sera shown to contain P. falciparum antibodies by RIA (Zavala et al., 1986). A commercial ELISA kit that uses NANP as the antigen has also been developed (Esposito et al., 1990). Other Assays Assays that detect antibodies are generally easier to develop than those that detect antigens. There are a number of potentially simple, inexpensive antigen-based assay systems that may be used to diagnose malaria. These include latex bead agglutination, solid-phase dipstick, membrane dot-blot, and hemagglutination. At present, these tests offer generally lower sensitivity and cost in exchange for increased speed and convenience. Although in certain instances sensitivity may be less critical than speed and ease of use, a decision to use one of these tests as a replacement for microscopy should be given careful consideration. One of the more promising rapid diagnostic methods that should be evaluated for use in malaria is the autologous red blood cell agglutination assay (Kemp et al., 1988). The technology, developed for detecting antibodies to the AIDS virus, utilizes a functionally univalent antibody reagent that binds to but does not agglutinate human red blood cells. A selected monoclonal antibody or polyclonal antiserum is then chemically conjugated to a monoclonal antibody reagent. When mixed with whole blood, the monoclonal antibody conjugates bind to the red blood cells, which then agglutinate if antigen is present. This assay, potentially rapid, simple, and inexpensive, may also be used to detect serum antibodies in whole blood by conjugating the monoclonal antibody to a specific antigen. Specifications for Diagnostic Tests The needs of those who use diagnostic tests must be considered when new assays are developed. Table 5-1 summarizes a set of test specifications for various categories of users. Biotechnology companies, university

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MALARIA: Obstacles and Opportunities TABLE 5-1 Suggested Specifications for Malaria Diagnostic Tests   Level of Carea Desired Quality of Diagnostic Test 1 2 3 4 5 Identify specimen as positive or negative for malaria + + + + + Identify Plasmodium falciparum + + + + + Identify other human Plasmodium species − + + + + Estimate parasite density − + + + − Use whole blood + + − − − Use small sample volume (no venipuncture) + − − − + Be easily transportable + − − − + Not require electricity + + − − + Provide results in less than one hour + +b + + − Be simple to perform + + + + − Have long reagent shelf life at ambient temperatures + + +b − + Be sensitive +c +c +c +c + Be highly specific − + − − − Be inexpensive + + +d − − Be amenable to batch processing − − − − + Provide a simple means of specimen collection − − − − + Be easily labeled − − − − + Differentiate between asexual and sexual forms − − − − + Provide a quantitative readout (high/medium/low) − − − − + a 1 = Village level; 2 = health clinic; 3 = district hospital; 4 = central hospital; 5 = epidemiologic survey. b Refrigeration may be available at this level of care. c Important if nonimmune populations are being tested. d Of somewhat less importance at this level than at levels 1 and 2. researchers, and others contemplating the development of new malaria diagnostics or the improvement of existing tests may find these parameters useful. Evaluation of Diagnostic Tests A diagnostic test should correctly differentiate between individuals who are infected and those who are not. The validity of a diagnostic test is usually determined by its sensitivity and specificity (Table 5-2). Sensitivity indicates the probability that a test is positive, given that the individual tested is indeed infected. As the sensitivity of a test increases, the number of false negatives (individuals who are infected but test negative) will decrease. Specificity indicates the probability an individual will test negative, given that he or she does not have the disease. As the specificity of a test increases, the number of false positives (individuals incorrectly classified as being infected) will decrease. In short, a highly sensitive test is very good at identifying those who are infected; a highly specific test is very good at identifying those who are not infected.

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MALARIA: Obstacles and Opportunities TABLE 5-2 Two-by-Two Table for Determining Diagnostic Test Characteristics   Disease Status Category Positive Negative Total Test result Positive TP FP TP+FP Negative FN TN FN+TN Total TP+FN FP+TN N Abbreviations: TP, true positive; FP, false positive; FN, false negative; TN, true negative. Formulas for calculating test characteristics: Sensitivity (true-positive rate) = TP/(TP+FN) = P(T+/D+) Specificity (true-negative rate) = TN/(FP+TN) = P(T−/D−) Positive predictive value = PV+ = P(disease/test+) = TP/(TP+FP) Negative predictive value = PV− = P(no disease/test−) = TN/(FN+TN) Multiply answers from the formulas by 100 to obtain percent. In large epidemiologic surveys, it is more important to determine the probability that a person who tests positive is indeed infected than it is to know the sensitivity or specificity of an individual test. As illustrated in Table 5-3, the positive predictive value of a diagnostic test with a fixed sensitivity and specificity will vary according to the prevalence of a disease in a given population. Example: A diagnostic test with a sensitivity of 95 percent and a specificity of 90 percent is applied to 1,000 people in each of two areas. The prevalence of malaria infection is estimated to be 10 percent in area A (see Table 5-3, Example 1) and 50 percent in area B (see Table 5-3, Example 2). As can be seen, in test area A, where the prevalence of malaria is 10 percent, the chance of correctly detecting malaria infection (positive predictive value) with the assay in question is slightly better than 50 percent, while for area B it is over 90 percent. The probability that someone who tests negative is indeed malaria free (negative predictive value) is high for both areas. Clearly, knowledge of disease prevalence is crucial to assessing the predictive value of any diagnostic technique. (It is possible to increase a test's positive predictive value by boosting its specificity. For example, if the specificity of the diagnostic assay in Table 5-3 were increased to 97 percent, the positive predictive value would rise to 77.8 percent.) RESEARCH AGENDA In situations where skilled microscopists have access to functioning microscopes and reagents for preparing and staining blood films, microscopy

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MALARIA: Obstacles and Opportunities TABLE 5-3 Sample Determinations of Diagnostic Test Characteristics   Disease Statusa Category Positive Negative Total Example 1b Test result Positive 95 90 185 Result 5 810 815 Total 100 900 1,000 Example 2c Test result Positive 475 50 525 Negative 25 450 475 Total 500 500 1,000 a Example 1—disease prevalence is 10%; Example 2—disease prevalence is 50% b Sensitivity = 95/100 = 0.95 = 95%; Specificity = 810/900 = 0.90 = 90%; Positive predictive value = 95/185 = 0.514 = 51.4%; Negative predictive value = 810/815 = 0.994 = 99.4% c Sensitivity = 475/500 = 0.95 = 95%; Specificity = 450/500 = 0.90 = 90%; Positive predictive value = 475/525 = 0.905 = 90.5%; Negative predictive value = 450/475 = 0.947 = 94.7% is still the gold standard of malaria diagnosis. There are circumstances in which new diagnostic techniques would be useful, however. Health care workers in remote villages and poorly equipped clinics would benefit from simpler diagnostic tests. RESEARCH FOCUS: Development of parasite antigenor nucleic acid-based detection methods that use finger-prick blood samples and require no electricity and no complicated or expensive equipment. Health care workers in nonendemic areas, where malaria occurs primarily in nonimmune individuals who become symptomatic at very low levels of parasitemia, would benefit from diagnostic methods that do not require parasites to be identified by microscopic morphology. RESEARCH FOCUS: Development of parasite antigenor nucleic acid-based diagnostics with limits of detection equivalent to or better than those of standard microscopy and that rely on clear-cut instrument interpretation or readouts.

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MALARIA: Obstacles and Opportunities Epidemiologists trying to determine the prevalence of malaria infection in a population would benefit from diagnostic tests that are less labor-intensive than standard microscopy. RESEARCH FOCUS: Development of parasite antigenor nucleic acid-based detection methods that collect finger-prick blood samples for later batch processing and that are inexpensive and easy to perform. REFERENCES Avidor, B., J. Golenser, and D. Sulitzeanu. 1985. Detection of Plasmodium falciparum using a radioimmunoassay based on a crossreacting, monoclonal anti-P. berghei antibody-P. berghei antigen system. Journal of Immunological Methods 82:121-129. Avraham, H., J. Golenser, D. Bunnag, P. Suntharasamai, S. Tharavanij, K. T. Harinasuta, D. T. Spira, and D. Sulitzeanu. 1983. Preliminary field trial of a radioimmunoassay for the diagnosis of malaria. American Journal of Tropical Medicine and Hygiene 32:11-18. Barker, R. H. Jr., L. Suebsaeng, W. Rooney, G. C. Alecrim, H. V. Duorado, and D. F. Wirth. 1986. Specific DNA probe for the diagnosis of Plasmodium falciparum malaria. Science 231:1434-1436. Barker, R. H. Jr., A. D. Brandling-Bennett, D. K. Koech, M. Mugambi, B. Khan, R. David, J. R. David, and D. F. Wirth. 1989a. Plasmodium falciparum: DNA probe diagnosis of malaria in Kenya. Experimental Parasitology 69:226-233. Barker, R. H. Jr., L. Suebsaeng, W. Rooney, and D. F. Wirth. 1989b. Detection of Plasmodium falciparum infection in human patients: a comparison of the DNA probe method to microscopic diagnosis. American Journal of Tropical Medicine and Hygiene 41:266-272. Bein, K., and P. Olcen. 1984. Detection of malaria antigens in urine using a solid-phase radioimmunoassay: preliminary study. Ethiopian Medical Journal 22:119-127. Bruce-Chwatt, L. J. 1987. From Laveran's discovery to DNA probes: new trends in diagnosis of malaria. Lancet 2:1509-1511. Del Giudice, G., A. S. Verdini, M. Pinori, A. Pessi, J.-P. Verhave, C. Tougne, B. Ivanoff, P.-H. Lambert, and H. D. Engers. 1987. Detection of human antibodies against Plasmodium falciparum sporozoites using synthetic peptides. Journal of Clinical Microbiology 25:91-96. Demedts, P., C. Vermuelen-Van Overmeir, and M. Wery. 1987. Simultaneous use of Plasmodium falciparum crude antigen and red blood cell control antigen in the enzyme-linked immunosorbent assay for malaria. American Journal of Tropical Medicine and Hygiene 36:257-263. Esposito, F., P. Fabrizi, A. Provvedi, P. Tarli, A. Habluetzel, and S. Lombardi. 1990. Evaluation of an ELISA kit for epidemiological detection of antibodies to Plasmodium falciparum sporozoites in human sera and bloodspot eluates. Acta Tropica 47:1-10.

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