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1 CE update [microbiology molecular diagnostics] Rapid Diagnosis of Malaria Bevinje Srinivas Kakkilaya, MD K.S. Hegde Medical Academy, Deralakatte, India DOI: /J4ANKCCJ147JB2FR After reading this article, the reader should understand how an immunochromatographic test works in diagnosing malaria. The reader should also understand how this test compares to the detection of malaria via the blood smear. Microbiology exam questions and the corresponding answer form are located after the Your Lab Focus section on p In the last century, peripheral blood smear examination has been the gold standard for the diagnosis of malaria, providing the most comprehensive information on a single test format. The immunochromatographic tests for the detection of malaria antigens, developed in the past decade, have opened a new and exciting avenue in malaria diagnosis; however, their role in the management and control of malaria appears to be limited at present. Malaria affects more than 300 million and kills at least 1 million people every year. 1 Early diagnosis and prompt treatment is the key to minimize the morbidity and mortality due to malaria, but the diagnostic capabilities for malaria remain a challenge for the laboratories and a hindrance for effective malaria control. 2,3 The quest for newer and easier diagnostic methods for malaria has picked up momentum in the last 10 years; and the development of the rapid diagnostic tests (RDTs) has opened a new avenue, calling for a review of the existing methods. 2,3 The factors that can have a bearing on the identification and interpretation of malaria parasitemia on a diagnostic test include the different malaria species; the different stages of erythrocytic schizogony; the endemicity of different species; the population movements; the interrelation between the levels of transmission, immunity, parasitemia, and the symptoms; the problems of recurrent malaria, drug resistance, persisting viable or non-viable parasitemia, and sequestration of the parasites in the deeper tissues; and the use of chemoprophylaxis or even presumptive treatment on the basis of clinical diagnosis. Microscopic Tests For nearly a hundred years, the direct microscopic visualization of the parasite on the thick and/or thin blood smears has been the accepted method for the diagnosis of malaria in most settings, from the clinical laboratory to the field surveys. The careful examination of a well-prepared and well-stained blood film currently remains the gold standard for malaria diagnosis. 2,3 The peripheral blood smear provides comprehensive information on the species, the stages, and the density of parasitemia with a sensitivity of 5 to 10 parasites/µl of blood for an experienced laboratory professional. 2 The efficiency of the test depends on the quality of the equipment and reagents, the type and quality of the smear, skill of the technician, the parasite density, and the time spent on reading the smear. 3,4 The test takes about 20 to 60 minutes depending on the proximity of the laboratory and other factors mentioned above. It is estimated to cost about 12 to 40 cents per slide in the endemic countries. 2 Non-Microscopic Tests Increasing global malaria mortality, the general lack of progress in malaria control, and the deficiencies of light microscopy and clinical diagnosis have spurred the need to develop simple and cost-effective diagnostic tests for malaria. Several attempts have been made to take the malaria diagnosis out of the realm of the microscope and the microscopist. Nucleic acid probes and immunofluorescence for the detection of Plasmodia within the erythrocytes; geldiffusion, counter-immunoelectrophoresis, radio immunoassay, and enzyme immunoassay for malaria antigens in the body fluids; and hemagglutination test, indirect immunofluorescence, enzyme immunoassay, immunochromatography, and Western blotting for anti-plasmodial antibodies in the serum have all been developed. 5-7 These tests have found some limited applications in research, retrograde confirmation of malaria, investigation of cryptic malaria, transfusion blood screening, and investigation of transfusion acquired infection. 5 In travelers returning to developed countries, studies based on polymerase chain reaction (PCR) have been found to be highly sensitive and specific for detecting all 4 species of malaria, particularly in cases of low level parasitemia and mixed infections The PCR test is reportedly 10-fold more sensitive than microscopy, 9 with one study reporting a sensitivity to detect 1.35 to 0.38 parasites/µl for P. falciparum and 0.12 parasites/µl for P. vivax. 10 The PCR test has also been found useful in unraveling the diagnosis of malaria in cases of undiagnosed fever. 11 In this context, the immunochromatographic tests for the qualitative detection of malaria antigens have generated great interest. 2,12 Intending to cut the time and effort needed for the diagnosis of malaria,

2 these RDTs do not require a laboratory, electricity, or any special equipment. 2,13 Immunochromatographic Tests for Malaria Antigens Immunochromatographic tests are based on the capture of the parasite antigens from the peripheral blood using either monoclonal or polyclonal antibodies against the parasite antigen targets. 2,3 Currently, immunochromatographic tests can target the histidine-rich protein 2 of P. falciparum, 3,14,15 a pan-malarial Plasmodium aldolase, 3,16 and the parasitespecific lactate dehydrogenase. 3,17 Histidine-rich protein 2 of P. falciparum (PfHRP2) is a water soluble protein that is produced by the asexual stages and gametocytes of P. falciparum, expressed on the red cell membrane surface, and shown to remain in the blood for at least 28 days after the initiation of antimalarial therapy Several RDTs targeting PfHRP2 have been developed. 2,3 Plasmodium aldolase is an enzyme of the parasite glycolytic pathway expressed by the blood stages of P. falciparum as well as the non-falciparum malaria parasites. 16,21 Monoclonal antibodies against Plasmodium aldolase are pan-specific in their reaction and have been used in a combined P.f/P.v immunochromatographic test that targets the pan malarial antigen (PMA) along with PfHRP2. 3,21 Parasite lactate dehydrogenase (pldh) is a soluble glycolytic enzyme produced by the asexual and sexual stages of the live parasites and it is present in and released from the parasite infected erythrocytes. 3,22 It has been found in all 4 human malaria species, and different isomers of pldh for each of the 4 species exist. 3 With pldh as the target, a quantitative immunocapture assay, a qualitative immunochromatographic dipstick assay using monoclonal antibodies, an immunodot assay, and a dipstick assay using polyclonal antibodies have all been developed The RDTs have been developed in different test formats like the dipstick, strip, card, pad, well, or cassette; and the latter has provided a more satisfactory device for safety and manipulation. 2,3 The test procedure varies between the test kits. In general, the blood specimen (2 to 50 µl) is either a finger-prick blood specimen, anticoagulated blood, or plasma, and it is mixed with a buffer solution that contains a hemolyzing compound and a specific antibody that is labeled with a visually detectable marker such as colloidal gold. In some kits, labeled antibody is predeposited during manufacture and only a lysing/washing buffer is added. If the target antigen is present in the blood, a labeled antigen/antibody complex is formed and it migrates up the test strip to be captured by the pre-deposited capture antibodies specific against the antigens and against the labeled antibody (as a procedural control). 2 A washing buffer is then added to remove the hemoglobin and permit visualization of any colored lines formed by the immobilized antigen-antibody complexes. 2 The pldh test is formatted to detect a parasitemia of >100 to 200 parasites/µl 23,25 and some of the PfHRP2 tests are said to detect asexual parasitemia of >40 parasites/µl. 2,7 The PfHRP2 test strips have 2 lines, 1 for the control and the other for the PfHRP2 antigen. The PfHRP2/PMA test Sample Origin Anti-falciparum antibody Anti-malaria antibody (all species) Control antibody Absorbent pad Components of Rapid Malaria Test Negative [F1] Rapid diagnostic test format and example results. Example Results strips and the pldh test strips have 3 lines, 1 for control, and the other 2 for P. falciparum (PfHRP2 or pldh specific for P. falciparum) and non-falciparum antigens (PMA or pan specific pldh), respectively. Change of color on the control line is necessary to validate the test and its non-appearance, with or without color changes on the test lines, invalidates the test. With color change only on the control line and without color change on the other lines, the test is interpreted as negative. With the PfHRP2 test, color change on both the lines is interpreted as a positive test for P. falciparum malaria. With the PfHRP2/PMA and the pldh tests, color change on the control line and the pan specific line indicates non-falciparum infection and color change on all the 3 lines indicates the presence of P. falciparum infection, either as monoinfection or as a mixed infection with nonfalciparum species [F1]. 2,3,21,25-27 Also, if the PfHRP2 line is visible when the PMA line is not, the test is interpreted as positive for P. falciparum infection. 21,28 Mixed infections of P. falciparum with the non-falciparum species cannot be differentiated from pure P. falciparum infec- Nonfalciparum malaria Pure or mixed infection with P. falciparum Invalid 603

3 tions. 29,30 However, with regard to the pldh test, it is claimed that in the presence of P. vivax infection, the genus specific line is much darker and more intense than the species specific line due to the presence of all the stages of the parasite in the blood. 26 Rapid diagnostic tests can be performed by individuals with minimal training. With the different tests that are currently available, the procedure may involve 2 to 6 steps and take 5 to 30 minutes. 2,13 The cost of the RDT also varies from test to test and from country to country, ranging from $1.20 to $13.50 per test. 13 Several RDTs have been evaluated in different settings, in comparison with microscopy, fluorescence microscopy, and PCR; and they already have a strong market presence. 2,3 However, none of the RDTs have been approved by the Food and Drug Administration for the diagnosis of malaria in the United States. 31 Because sufficient information is not available on the performance of the malaria antigen detection devices and because they are of substantial importance in diagnosing and preventing life-threatening illness, the Center for Devices and Radiological Health has classified these devices as Class III and a premarket approval application is required. 32 Cross-Reactions With Autoantibodies Studies have reported cross reactivity of the various RDTs with autoantibodies such as rheumatoid factor, resulting in false positive tests for malaria. In a study of 173 travelers returning from malaria endemic areas, the PfHRP2 test was true positive in 26 patients and false positive in 4 patients; 3 of these patients had rheumatoid factor and the fourth had low titers of antinuclear antibodies. 33 Studies in patients with positive rheumatoid factor have shown that the false positive reactions are higher with the PfHRP2 tests using IgG capture antibody (16.5% to 83%) compared to the PfHRP2 tests using IgM antibodies (6.6%) 36 and the pldh test (3.3%). 36,37 Cross reactivity of the PMA antibody with rheumatoid factor does not appear to occur. 21 Comparative Studies of RDTs For the diagnosis of P. falciparum malaria, some studies comparing the PfHRP2 and pldh tests have found relatively higher sensitivity with the PfHRP2 tests and higher specificity with the pldh test, while some others have found a higher sensitivity with the pldh test. 12,26,29,38-43 In a field study comparing 10 different RDTs, the parasitemia for 50% detection limit was found to be lower for the PfHRP2 tests compared to the pldh test and false positive results on day 7 after treatment were fewer with the pldh test. 44 A study comparing 5 different PfHRP2 tests found considerable differences among the tests. 45 For the diagnosis of P. vivax infection, the pldh test is found to be more sensitive and specific than the PMA test. 40,46 A comparison is given in T1. In a study from a holoendemic area, a positive pldh test was found to be a better indicator of malaria than a positive PfHRP2/PMA test, and the diagnostic reliability of a positive pldh test approached that of an ideal test. 39 RDTs in Field Studies The RDTs have been extensively field tested in the areas of different endemicity and different levels of health care facilities, including the remote villages and forests. Compared to microscopy, the PfHRP2/PMA tests were found to be less sensitive in detecting asymptomatic patients, particularly at low parasitemias. 21 The sensitivity of the pldh test in field studies was also found Comparison of Rapid Diagnostic Tests for Malaria Antigens PfHRP2 tests PfHRP2 and PMA test pldh test T1 604 Target antigen Histidine rich protein 2 of P. falci- Pan-specific Plasmodium aldolase, Parasite lactate dehydrogenase, parum, water soluble protein parasite glycolytic enzyme parasite glycolytic enzyme produced expressed on RBC membrane produced by all species and by all species PfHRP2 General test format 2 lines 3 lines 3 lines Capability Detects P. falciparum only Can detect all 4 species Can detect all 4 species Non-falciparum species Not detected Detected; differentiation between Detected; differentiation between the the 3 not possible 3 not possible Mixed infections of P. falciparum with Appear as P. falciparum; Appear as P. falciparum; Appear as P. falciparum; differentiation non-falciparum species differentiation not possible differentiation not possible not possible Detection limit > parasites/µl Higher for P. vivax and other non- > parasites/µl for P. falciparum falciparum species and P. vivax; may be higher for P. malariae and P. ovale Post-treatment persistence of antigens Reported up to 31 days Reported; longer for pan specific Reported up to 1-3 weeks antigenemia than for PfHRP2 Cross-reactivity between malarial Reported Reported Reported species Cross-reactivity with auto antibodies Reported, high (up to 83% with Not known Reported, low (3.3% with rheumatoid rheumatoid factor) factor) Indication of viability of parasites No No Positive test indicates presence of viable parasitemia

4 Comparison of Peripheral Blood Smear Examination and Rapid Diagnostic Tests for Malaria Peripheral Smear Rapid Diagnostic Tests T2 Format Slides with blood smear Test strip Equipment Microscope Kit only Training Trained microscopist Anyone with a little training Test duration minutes or more 5-30 minutes Test result Direct visualization of the parasites Color changes on antibody coated lines Capability Detects and differentiates all plasmodia at Detects malaria antigens (PfHRP2/ PMA/ pldh) from different stages asexual and/or sexual forms of the parasite Detection threshold 5-10 parasites/µl of blood /µL for P. falciparum, higher for non-falciparum Species differentiation Possible Cannot differentiate among non-falciparum species; mixed infections of P. falciparum and non-falciparum appear as P. falciparum Quantification Possible Not possible Differentiation between sexual Possible Not possible and asexual stages Disadvantages Availability of equipment and skilled microscopists, Unpredictable efficiency at low and very high parasitemia; particularly at remote areas and odd hours cross reactions among plasmodial species and with auto-antibodies; persistence of antigens Status Gold standard Not yet approved by the FDA Cost per test US$ US$ to be lower at low parasitemias in field studies. 30 The comparisons between the PfHRP2/PMA test and the pldh test in field studies have yielded variable results, but the pldh tests were found to have a better specificity for P. vivax. 40 In one study, PfHRP2, PfHRP2/PMA, and pldh tests had a sensitivity of <75% at parasitemias of <1,000/µL. 40 However, some studies, particularly at the remote forests and villages, have found the RDTs to be useful and easy tools for field surveys, because they are easily read by the field workers without supervision and require no training or instruments Some studies have found the experience and the level of training of the field staff can influence the sensitivity and specificity of these tests, 50 and have reported questionable results or failure to interpret the results in 1.7% to 3.75% of the PfHRP2/PMA test strips. 51,52 RDTs for the Diagnosis of Malaria in Travelers The RDTs have been evaluated for the diagnosis of malaria in travelers, as self-use kits and at the laboratories. For P. falciparum malaria, the sensitivity was found to be 82% to 94% with the PfHRP2 tests and 83% to 89% with the pldh test; for P. vivax malaria, the pldh test had a sensitivity of 61.5%. 12,29,38,42 With the PfHRP2/PMA test, the sensitivity for the diagnosis of P. falciparum and P. vivax infection were 87.8% and 76.5%, respectively. 53 However, another study showed a high degree of variability with species other than P. falciparum or when there were more than 1 Plasmodium species. 54 Studies on self-use by travelers have raised doubts over the reliability of interpretation of the RDTs by travelers. In one study, only 68% of the European tourists to Kenya were able to perform the test correctly, and 10 out of 11 with malaria failed to diagnose themselves correctly. 38,55 High number of false negative results have also been reported. 38,56 Written instructions supplemented with verbal instructions improved the sensitivity to 90% compared to 75% with written instructions alone. 38 Conclusions Diagnosis of malaria disease is more than the mere detection of malaria infection. While the demonstration of the malaria parasites or antigens in the peripheral blood indicates malaria infection, the diagnosis of malaria disease and the decisions on its treatment would depend on the various host factors and the endemicity of the infection. If one cannot correctly interpret the presence of parasites in the blood of the patient, parasitological diagnosis of malaria is not useful. In this context, a test that provides comprehensive information on the species, the stages, and the severity of parasitemia is more useful and dependable. For a century or more, the peripheral blood smear catered to these diverse needs. The RDT kits are relatively new and can be carried in travel bags, kept in the clinicians desks, or even made available in remote forests and villages. The RDTs do not require any laboratory equipment or electricity. It is claimed that with the RDTs, the diagnosis of malaria can be taken to the doorsteps of the patients and the results can be seen by the patients themselves for better acceptance and treatment compliance. 2,3 As compared to the blood smear examination [T2], the RDTs can be performed easily in 5 to 30 minutes by the field workers, untrained personnel, travelers, or others with a minimum training of 3 to 24 hours. 2,3 It has been suggested that the RDTs can be useful in areas where specialized laboratories or even microscopy are unavailable and when urgent malaria diagnosis is needed by a practitioner without the delay associated with the laboratory. The joint WHO/USAID informal consultation held in October 1999 concluded that the RDTs should provide results at least as accurate as those derived from microscopy performed by an average technician under routine field conditions. 2 For that purpose, the RDTs should be able to detect all 4 species of malaria parasite and differentiate P. falciparum 605

5 606 from the other species. The overall sensitivity (compared to expert microscopy) should be greater than 95%. Parasite densities greater than 100 asexual parasites per µl blood should be detected reliably with sensitivity close to 100%, and the specificity should be at least 90% for all malaria species. 2 According to the WHO/USAID consensus report, RDTs for the diagnosis of P. falciparum malaria generally achieve a sensitivity of >90% at densities above 100 parasites per µl blood and the sensitivity decreases markedly below that level of parasite density. 2 Many studies have achieved >95% sensitivity at parasitemia of 500 parasites/µl, but this high parasitemia is seen in only a minority of patients. 30,51,57,58 For the diagnosis of P. vivax malaria, the PfHRP2/PMA test has a lower sensitivity compared to that for P. falciparum malaria; however, the pldh test has an equal or better sensitivity for P. vivax malaria compared to P. falciparum malaria. 23,26,40,43,46,51 For the diagnosis of P. malariae and P. ovale infections, the sensitivity is lower than that of P. falciparum malaria at all levels of parasitemia on both the PfHRP2/PMA and the pldh tests. 22,28,53 The specificity appears to be better with the pldh test than the PfHRP2/PMA test for both P. falciparum and non-falciparum malaria. 12,38-42 The sensitivity of the RDTs at low levels of parasitemia and for non-immune populations remains a problem. 2,3 Of concern is the fact that in nonimmune individuals, symptomatic malaria can occur at parasite densities that are below the detection threshold of currently available RDTs. 2 In a cross-sectional malaria survey, 84.1% patients with P. falciparum infection had a parasitemia of <500/µL and the sensitivity of the PfHRP2 test was only 23.3% at this level of parasitemia. 51 The level of parasitemia encountered in P. vivax infection rarely exceeds 1% (50,000/µL) and usually is much lower. The level of parasitemia for P. malariae and P. ovale are lower than for P. vivax, and the affinity of the panspecific antibodies for these parasites is also lower. 3 The nonimmune subjects have a lower pyrogenic threshold. In a large series of nonimmune subjects, more than 70% of subjects developed fever at parasite densities of <100/µL. 59 The pyrogenic threshold is also lower for P. vivax malaria compared to P. falciparum malaria. 59 Lower levels of parasitemia are also common in nonimmune patients treated with antimalarial chemoprophylaxis. 8 This would mean that P. falciparum infections with low levels of parasitemia and a significant proportion of symptomatic, non-immune patients with P. vivax (or other non-falciparum) malaria may be missed by the RDTs. 21,51 Because of this limited usefulness in detecting low-level parasitemia (<100 parasites/µl), the use of RDTs is not indicated for diagnosing infection in most clinical presentations of malaria in the United States. Further, the RDTs have been reported to give false negative results even at higher levels of parasitemia. 13 Therefore, in cases of suspected severe malaria or complex health emergencies, a positive result may be confirmatory but a negative result may not rule out malaria. Further, a negative RDT result should always be confirmed by microscopy. 2 It should be emphasized that P. falciparum malaria, a potentially lethal disease, must not be missed because of a false negative dipstick test. 12 It has been suggested that in such cases, 1 in 10 dilution of a negative sample with 0.9% sodium chloride may help to exclude the prozone phenomenon. The performance of the RDTs is reported to be influenced by a multitude of factors like the type of the parasite and the level of parasitemia; the type of test; the target antigen and the capture antibody; the expression of the target antigens on the parasites and the presence of several isomers; the presence of gametocytemia; persistent antigenemia or sequestration of the parasites; cross-reactions with other malaria species and with autoantibodies; batch quality variations in test strips; prozone phenomenon; and prior treatment. 2,3,8,12,20,21,30,45,51 The interpretation of the color changes to identify the malaria infection is influenced by the level of training, the type of instructions, and in case of self-use, by the state of the patient. 38,50 The performance on self-use of RDTs by the sick or delirious patients may be impaired, and it has been suggested that a healthy companion should be asked to do the test. Thus, the sensitivity and specificity of the RDTs, and hence the diagnosis and treatment of malaria based on the RDTs, are influenced by the positive results due to causes other than malaria antigenemia, and the negative results due to causes other than low parasitemia. Another major difficulty still encountered by the use of RDTs is the correct identification of Plasmodium species, particularly in areas where nonfalciparum malaria is prevalent. 2 With reports of the RDTs detecting all 4 malaria species, a brand name like P.f./P.v. test can be confusing. Also while using the PfHRP2 tests, it should be kept in mind that these tests can detect only P. falciparum infection and would miss the more common non-falciparum malaria in areas where other Plasmodium species are co-endemic. These tests are not suitable for diagnosing the cases of imported malaria from areas where P. falciparum is not necessarily the most prevalent species. 2,26 Also, the inability to quantify and differentiate between the sexual and asexual parasitemia could pose problems in the areas of high transmission and in cases of incomplete treatment. Therefore, the identification of the color changes on the RDT strips may look simple but the interpretation of the result would require the knowledge of the malarial dynamics and of the possible errors with the RDTs. Otherwise, the RDTs may raise more questions than answers, and the insufficient accuracy of the RDTs could increase the number of incorrect malaria diagnoses. 8 Lower sensitivity in detecting asymptomatic patients, large numbers of positive tests due to persistent antigenemia following incomplete treatment, inability to differentiate the mixed infections and the non-falciparum species, and inability to differentiate between the various stages of the parasite limit the value of the RDTs in active surveillance in the field. The cost of the RDTs has also been considered as a

6 major obstacle for their large scale use in field studies. Stand-by emergency self-treatment in travelers has been proposed as another application for RDTs and kits for selfdiagnosis (some also for self-treatment, with antimalarial drugs) are marketed in several European countries. 2 Low levels of sensitivity of RDTs, high degree of variability with non-falcipaum and mixed infections, problems in interpretation of the results by the travelers with unacceptably high false negative interpretations are all limitations for the use of RDTs in malaria diagnosis in travelers. The RDT kits may be recommended for self-use by travelers only after major technical modifications. 3 Appropriate instruction and training should include successful performance of the test procedure. 2,38 While the therapeutic response is assessed by the decrease in or disappearance of asexual parasitemia, all the antigens targeted by the RDTs are expressed by the asexual as well as the sexual forms of the parasites. 2,3 Further, except for the artemisinin compounds, the schizonticidal drugs have no effect on the gametocytes of P. falciparum. 18 Occurrence of positive results due to persistent antigenemia following treatment constrains the assessment of treatment failure and follow-up. 2,18 Therefore, the predictive value of the RDTs in assessing the response to therapy and the recrudescence appears limited. In one study, the PPV of the PfHRP2 and PMA test for treatment failure were 76.9% and 87% on day 3 and 78.9% and 78.9% on day 14, respectively. 60 The negative results were found to be even less predictive of adequate response, with false negative results in onesixth of patients who developed recrudescence by microscopy. 60 To reliably predict treatment outcome with RDTs, further development appears necessary to improve the sensitivity for viable parasites while avoiding detection of both gametocytes and persistent antigen in convalescence. 60 Blood film microscopy appears to be the best choice at present for this purpose. 5 Because pldh is produced only by the live parasites, treated infections and dead parasites are expected to give negative results. Therefore, the pldh test can thus differentiate living parasites from dead ones. 22,26 But used alone, the pldh test may be negative in dead infections and as such cannot make such a differentiation. Therefore, the RDTs as stand-alone tests have a limited use in the diagnosis and follow-up of malaria. The joint WHO/USAID informal consultation held in October 1999 arrived at a consensus on the use of RDTs. 2 In regions where the prevalence is fairly uniform and asymptomatic parasitemia is common, the RDTs are unlikely to be of any value and clinical diagnosis alone is incorporated in malaria treatment guidelines; however, the RDTs may be used in peripheral locations where microscopy is not available, in suspected severe malaria and to confirm treatment failure. 2 The RDTs would be most useful in areas where there is low or moderate transmission of malaria, especially in the remote regions where microscopy is not available and where multi-drug resistance, requiring use of expensive drugs, is common. 2 Other suitable settings for the use of RDTs may include suspected malaria epidemics, complex health emergencies, care of nonimmune persons and confirmation of severe malaria. 2 However, in all doubtful cases, peripheral blood smear should be examined. It can be said that the RDTs have opened a new and exciting avenue in the diagnosis and control efforts of malaria. Strengthening this advantage depends on the future enhancements in the performance and the efficiency of the RDTs. This will occur by improving the sensitivity and specificity, minimizing or nullifying the cross-reactions between species and with auto-antibodies, improving the ability to reflect viable asexual parasitemia, minimizing or nullifying the reactions with gametocytes, developing tests that can identify malaria as a certain cause of fever, identifying the potential markers for complications and treatment outcomes, and quantifying parasitemia. 2 Only after the refinement of RDTs as accurate diagnostic tools can they be considered for a wider role in the management and control of malaria. Until then, the RDTs may not be able to replace the peripheral smear examination as the most comprehensive and cost-effective test for malaria. 1. What is malaria? Available at: Sheet01.pdf. Accessed on December 1, New perspectives in malaria diagnosis: Report of a joint WHO/USAID informal consultation October, 1999 WHO 2000 Document No WHO/CDS/RBM/ ;WHO/MAL/ Moody A. Rapid diagnostic tests for malaria parasites. Clin Microbiol Review. 2002;15: Tham JM, Lee SH, Tan TMC, et al. Detection and species determination of malaria parasites by PCR: Comparison with microscopy and with ParaSight F and ICT Malaria Pf tests in a clinical environment. J Clin Microbiol. 1999;37: Thakor HG. Laboratory diagnosis of malaria. J Ind Med Assoc. 2000;98: Ray K. Immunodiagnosis of malaria. J Commun Dis. 1984;16: Afzaal S, Singh M, Fatima S, et al. Rapid diagnostic tests for malaria. J Assn Physicians India. 2001;49: Rubio JM, Buhigas I, Subirats M, et al. Limited level of accuracy provided by available rapid diagnosis tests for malaria enhances the need for PCR-based reference laboratories. J Clin Microbiol. 2001;39: Zhong KJY, Kain KC. Evaluation of a colorimetric PCR-based assay to diagnose Plasmodium falciparum malaria in travelers. J Clin Microbiol. 1999;37: Myjak P, Nahorski W, Pieniazek NJ, et al. Usefulness of PCR for diagnosis of imported malaria in Poland. Eur J Clin Microbiol Infect Dis. 2002;21: Vinetz JM, Li J, McCutchan TF, et al. Plasmodium malariae infection in an asymptomatic 74-year-old Greek woman with splenomegaly. N Eng J Med. 1998;338: Jelinek T, Grobusch MP, Schwenke S, et al. Sensitivity and specificity of dipstick tests for rapid diagnosis of malaria in non immune travelers. J Clin Microbiol. 1999;37: Singh N, Shukla M. An assessment of the usefulness of a rapid immunochromatographic test Determine TM malaria Pf in evaluation of intervention measures in forest villages of central India. BMC Infect Dis. 2001;1: Shiff CJ, Premji Z, Minjas ZN. The rapid manual Parasight-F test. A new diagnostic tool for Plasmodium falciparum infection. Trans R Soc Trop Med Hyg. 1993;87: Rock EP, Marsh K, Saul SJ, et al. Comparative analysis of the Plasmodium falciparum histidinerich proteins HRP1, HRP2 and HRP3 in malaria diagnosis of diverse origin. Parasitology. 1987;95: Meier B, Dobeli H, Certa U. Stage-specific expression of aldolase isoenzymes in the rodent malaria parasite Plasmodium berghei. Mol Biochem Parasitol. 1992;52: Makler MT, Hinrichs DJ. Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitemia. Am J Trop Med Hyg. 1993;48:

7 Tjitra E, Suprianto S, McBroom J, et al. Persistent ICT Malaria P.f./P.v Panmalarial and HRP2 antigen reactivity after treatment of Plasmodium falciparum malaria is associated with gametocytemia and results in false-positive diagnoses of Plasmodium vivax in convalescence. J Clin Microbiol. 2001;39: Verle P, Binh LN, Lieu TT, et al. ParaSight F test to diagnose malaria in hypoendemic and epidemic prone regions of Vietnam. Trop Med Int Health. 1996;1: Humar A, Ohrt C, Harrington MA, et al. Parasight F test compared with the polymerase chain reaction and microscopy for the diagnosis of Plasmodium falciparum malaria in travelers. Am J Trop Med Hyg. 1997;56: Tjitra E, Suprianto S, Dyer M, et al. Field evaluation of the ICT malaria P.f/P.v immunochromatographic test for detection of Plasmodium falciparum and Plasmodium vivax in patients with a presumptive clinical diagnosis of malaria in eastern Indonesia. J Clin Microbiol. 1999;37: Aslan G, Ulukanligil M, Seyrek A, et al. Diagnostic performance characteristics of rapid dipstick test for Plasmodium vivax malaria. Mem Inst Oswaldo Cruz. 2001;96: Piper RC, Le Bras J, Wentworth L, et al. Immunocapture diagnostic assays for malaria utilizing Plasmodium lactate dehydrogenase (pldh). Am J Trop Med Hyg. 1999;60: Kaushal DC, Kaushal NA. Diagnosis of malaria by detection of plasmodial lactate dehydrogenase with an immunodot enzyme assay. Immunol Invest. 2002;31: Standard Diagnostics. One step malaria P.f/P.v antigen rapid test: SD bioline malaria antigen. Available at: rapid/malaria_ag.htm. Accessed November 20, Palmer CJ, Lindo JF, Klaskala WI, et al. Evaluation of the OptiMAL test for rapid diagnosis of Plasmodium vivax and Plasmodium falciparum malaria. J Clin Microbiol. 1998;36: Pinto MJW, Pereira NF, Rodrigues S, et al. Rapid diagnosis of falciparum malaria by detection of Plasmodium falciparum HRP-2 antigen. J Assn Physicians India. 1999;47: Win TT, Tantular IS, Pusarawati S, et al. Detection of Plasmodium ovale by the ICT malaria P.f/P.v. immunochromatographic test. Acta Trop. 2001;80: Iqbal J, Hira PR, Sher A, et al. Diagnosis of imported malaria by plasmodium lactate dehydrogenase (pldh) and histidine-rich protein 2 (PfHRP-2)-based immunocapture assays. Am J Trop Med Hyg. 2001;64: Quintana M, Piper R, Boling HL, et al. Malaria diagnosis by dipstick assay in a Honduran population with coendemic Plasmodium falciparum and Plasmodium vivax. Am J Trop Med Hyg. 1998;59: Detection of parasite antigens. In diagnostic procedures: Diagnostic procedures for blood specimens. Available at: ocedures.htm. Accessed October 5, Chiang Syin. Antigen/antibody testing for malaria informational. Available at: b1kx.pdf. Accessed March 14, Laferl H, Kandel K, Pichler H. False positive dipstick test for malaria. N Eng J Med Nov;337: Mishra B, Samantaray JC, Kumar A, et al. J Clin Microbiol. 1999;37: Bartoloni A, Sabatinelli G, Benucci M. Performance of two rapid tests for Plasmodium falciparum malaria in patients with rheumatoid factors. N Eng J Med. 1998;338: Grobusch MP, Jelinek T, Hänscheid T. False positivity of rapid antigen detection tests for diagnosis of Plasmodium falciparum malaria: Issue appears to be more complicated than presented. J Clin Microbiol. 1999;37: Grobusch MP, Alpermann U, Schwenke S, et al. False-positive rapid tests for malaria in patients with rheumatoid factor. Lancet. 1999;353: Jelinek T, Grobusch MP, Nothdurft HD. Use of dipstick tests for the rapid diagnosis of malaria in non immune travelers. J Travel Med. 2000;7: Tarimo DS, Minjas JN, Bygbjerg IC. Malaria diagnosis and treatment under the strategy of the integrated management of childhood illness (IMCI): Relevance of laboratory support from the rapid immunochromatographic tests ICT Malaria P.f./P.v. and OptiMal. Ann Trop Med Parasitol. 2001;95: Huong NM, Davis TM, Hewitt S, et al. Comparison of three antigen detection methods for diagnosis and therapeutic monitoring of malaria: A field study from southern Vietnam. Trop Med Int Health. 2002;7: Labbe AC, Pillai DR, Hongvangthong B, et al. The performance and utility of rapid diagnostic assays for Plasmodium falciparum malaria in a field setting in the Laos People s Democratic Republic. Ann Trop Med Parasitol. 2001;95: Ricci L, Viani I, Piccolo G, et al. Evaluation of optimal assay test to detect imported malaria in Italy. New Microbiol. 2000;23: Lee MA, Aw LT, Singh M. A comparison of antigen dipstick assays with polymerase chain reaction (PCR) technique and blood film examination in the rapid diagnosis of malaria. Ann Acad Med Singapore. 1999;28: Craig MH, Bredenkamp BL, Williams CH, et al. Field and laboratory comparative evaluation of ten rapid malaria diagnostic tests. Trans R Soc Trop Med Hyg. 2002;96: Guthmann JP, Ruiz A, Priotto G, et al. Validity, reliability and ease of use in the field of five rapid tests for the diagnosis of Plasmodium falciparum malaria in Uganda. Trans R Soc Trop Med Hyg. 2002;96: Cho D, Kim KII, Park SC, et al. Evaluation of rapid immunocapture assays for the diagnosis of Plasmodium vivax in Korea. Parasitol Res. 2001;87: Sharma SK, Tyagi PK, Haque MA, et al. Field studies on the sensitivity and specificity of an immunochromatographic test for the detection of Plasmodium falciparum malaria in tribal areas of Orissa. Indian J Malariol. 1999;36: Singh N, Valecha N. Evaluation of a rapid diagnostic test, Determine malaria pf, in epidemic-prone, forest villages of central India (Madhya Pradesh). Ann Trop Med Parasitol. 2000;94: Wolday D, Balcha F, Fessehaye G, et al. Field trial of the RTM dipstick method for the rapid diagnosis of malaria based on the detection of Plasmodium falciparum HRP-2 antigen in whole blood. Trop Doct. 2001;31: Min-Naing C, Gatton ML. Performance appraisal of rapid on-site malaria diagnosis (ICT Pf/Pv test) in relation to human resources at village level in Myanmar. Acta Trop. 2002;81: Coleman RE, Maneechai N, Rachapaew N, et al. Field evaluation of the ICT malaria Pf/Pv immunochromatographic test for the detection of asymptomatic malaria in a Plasmodium falciparum/vivax endemic area in Thailand. Am J Trop Med Hyg. 2002;66: Proux S, Hkirijareon L, Ngamngonkiri C, et al. Short communication. Parachek-Pf: A new, inexpensive and rapid test for P. falciparum malaria. Trop Med Int Health. 2001;6: Jelinek T, Grobusch MP, Harms G. Evaluation of a dipstick test for the rapid diagnosis of imported malaria among patients presenting within the network TropNetEurop. Scand J Infect Dis. 2001;33: Gatti S, Bernuzzi AM, Bisoffi Z, et al. Multicentre study, in patients with imported malaria, on the sensitivity and specificity of a dipstick test (ICT Malaria P.f./P.v.) compared with expert microscopy. Ann Trop Med Parasitol. 2002;96: Jelinek T, Amsler L, Grobusch MP, et al. Self-use of rapid tests for malaria diagnosis by tourists. Lancet. 1999;354: Funk M, Schlagenhauf P, Tschopp A, et al. MalaQuick versus ParaSight F as a diagnostic aid in travelers malaria. Trans R Soc Trop Med Hyg. 1999;93: Taylor WR, Widjaja H, Basri H, et al. Assessing the Parasight-F test in northeastern Papua, Indonesia, an area of mixed Plasmodium falciparum and Plasmodium vivax transmission. Am J Trop Med Hyg. 2002;66: Berchem NN, Leke RF, Tietche F, et al. Evaluation of a rapid test for histidine rich protein 2 for diagnosis of Plasmodium falciparum infection in Cameroonian children. Tran R Soc Trop Med Hyg. 1999;93: Kitchen S. Symptomatology: General considerations. In: Boyd M, ed. Malariology. Vol II. Philadelphia: W.B. Saunders. 1949; Tjitra E, Suprianto S, Dyer ME, et al. Detection of histidine rich protein 2 and panmalarial ICT Malaria Pf/Pv test antigens after chloroquine treatment of uncomplicated falciparum malaria does not reliably predict treatment outcome in eastern Indonesia. Am J Trop Med Hyg. 2001;65: