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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. 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%) and the pLDH test (3.3%). Cross reactivity of the PMA antibody with rheumatoid factor does not appear to occur.
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Sensitivity: 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. Many studies have achieved >95% sensitivity at parasitemia of ~500 parasites/µL, but this high parasitemia is seen in only a minority of patients. 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. vivaxmalaria compared to P. falciparum malaria. 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. The specificity appears to be better with the pLDH test than the PfHRP2/PMA test for both P. falciparum and non-falciparum malaria.
The sensitivity of the RDTs at low levels of parasitemia and for non-immune populations remains a problem. Compared to microscopy, the PfHRP2/PMA tests were found to be less sensitive in detecting asymptomatic patients, particularly at low parasitemias. The sensitivity of the pLDH test in field studies was also found to be lower at low parasitemias in field studies. 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. In one study, PfHRP2, PfHRP2/PMA, and pLDH tests had a sensitivity of <75% at parasitemias of <1,000/m L. Of concern is the fact that in non-immune individuals, symptomatic malaria can occur at parasite densities that are below the detection threshold of currently available RDTS. 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. 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. Lower levels of parasitemia are also common in non-immune patients treated with antimalarial chemoprophylaxis. 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.
Further, the RDTs have been reported to give false negative results even at higher levels of parasitemia. 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. It should be emphasized that P. falciparum malaria, a potentially lethal disease, must not be missed because of a false-negative dipstick test. 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.
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False Positivity: False positive tests can occur with RDTs for many reasons. Potential causes for PfHRP2 positivity, other than gametocytemia, include persistent viable asexual-stage parasitemia below the detection limit of microscopy (possibly due to drug resistance),persistence of antigens due to sequestration and incomplete treatment, delayed clearance of circulating antigen (free or in antigen-antibody complexes) and cross reaction with non-falciparum malaria or rheumatoid factor. Proportion of persistent positivity has been linked to the sensitivity of the test, type of test, degree of parasitemia and possibly the type of capture antibody.
False negativity: On the other hand, false negative tests have been observed even in severe malaria with parasitemias > 40000 parasites/µl. This has been attributed to possible genetic heterogeneity of PfHRP2 expression, deletion of HRP-2 gene, presence of blocking antibodies for PfHRP2 antigen or immune-complex formation, prozone phenomenon at high antigenemia or to unknown causes.
Cross reactions between Plasmodia species and problems in identifying non-falciparum species: Cross reaction of PfHRP2 with non-falciparum malaria could give false positive results for P. falciparumand mixed infections containing asexual stages of P. falciparum could be interpreted as negative in about one third of the patients.
Another major difficulty still encountered by the use of RDTs is the correct identification of Plasmodium species, particularly in areas where non-falciparum malaria is prevalent. The PfHRP2 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.
Multiple Influences: 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. 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. 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.
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. 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.
Persistence of antigens: All the antigens targeted by the RDTs are expressed by the asexual as well as the sexual forms of the parasites and persistent antigenemia can cause positive tests on RDTs up to one month. With the schizonticidal drugs having no effect on the gametocytes of P.falciparum (except for the artemisinin compounds), RDTs may not be reliable tools to predict the therapeutic response.
Interpretation: Although the RDTs have been reported to be useful and easy tools for field surveys in remote forests and villages, some studies have found that the experience and the level of training of the field staff can influence the sensitivity and specificity of these tests and have reported questionable results or failure to interpret the results in 1.7% to 3.75% of the PfHRP2/PMA test strips.
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 major obstacle for their large scale use in field studies.
The RDTs have been evaluated for the diagnosis of malaria in travelers, as self-use kits and at the laboratories. 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. High number of false negative results has also been reported.
A potential problem with the dipstick test is that the circulating antigen will be detectable for many days even after the elimination of viable P. falciparum from the blood stream. A positive test therefore may not always indicate an active infection. US FDA approves RDT: On June 13, 2007, the Food and Drug Administration (FDA) approved the first RDT for use in Africa and the United States. This RDT is approved for use by hospital and commercial laboratories, not by individual clinicians or by patients themselves. It is recommended that all RDTs are followed-up with microscopy to confirm the results and if positive, to quantify the proportion of red blood cells that are infected. Conclusion Ask for MP test in all cases of fever and related symptoms and also whenever there is high level of suspicion. MP test can be done at any time. Do not wait for typical symptoms and signs or for chills. A negative test does not rule out malaria. Repeated tests may have to be done in all doubtful cases. Duration of the illness, level of parasitemia, expertise of the Microscopist and the method of examination may all have a bearing on the result of the M.P. test.
The simplest and surest test is the time-honored peripheral smear study for malarial parasites. None of the other newer tests have surpassed the ‘gold standard’ peripheral smear study. This RDT is approved for use by hospital and commercial laboratories in remote areas (forests and villages), not by individual clinicians or by patients themselves.
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