Rapid Immunochromatography Test "ICT Malaria Pf" in Diagnosis of Plasmodium falciparum and its Application in the in vivo Drug Susceptibility Test

Gholam-Hossein Edrissian PharmD, Abbas Afshar, Gholam Mohsseni

School of Public Health and Institute of Public Health Research, Tehran University of Medical Sciences, Tehran, Iran

  • Abstract

    Background-Malaria is a main parasitic disease with high morbidity and mortality in the world. Rapid diagnosis and prompt treatment are the basic technical elements for the management and control of the disease. During the recent years rapid immunochromatography tests have been applied in diagnosis of specific antigens of human Plasmodia.
    Objective-The main objective of this study is the evaluation of immunochromatographic test for P. falciparum "ICT Malaria Pf" in the detection of parasitemia in falciparum malaria infection and also in the in vivo drug susceptibility test.
    Methods-Dipstick kits "ICT Malaria Pf" were applied in the diagnosis and in vivo assessment of the response of P. falciparum to chloroquine in patients with malaria in Bandar-Abbas, southeast Iran. The results of all tests were compared and evaluated with those of microscopic examination (ME) and parasite count in the stained thick blood smears.
    Results-ICT Malaria Pf was highly specific (reaching 100%) and sensitive (93%) in detection of P. falciparum parasitemia and also quite helpful in the in-vivo assessment of the response of the parasite to chloroquine.
    Conclusion- At present for the diagnosis of malaria in endemic countries, the dipstick kits are rather expensive. Nevertheless, it is very helpful in centers where the necessary facilities for ME are not accessible for the prompt diagnosis of falciparum infection.

  • Keywords • Falciparum malariarapid diagnosisimmunochromatography test

    Introduction

    Malaria is still the main parasitic disease with high morbidity and mortality. Despite the problem of drug resistance, malaria is a curable disease if the patients have access to early diagnosis and prompt treatment.1

    Rapid and accurate diagnosis is the key to effective disease management and one of the basic technical elements of the strategy for malaria control.2 So far the light microscopic examination (ME) of the stained blood smears have been considered as the standard gold test for the diagnosis of malaria. ME however, requires well trained and experienced malaria microscopists and it is also rather time consuming. Some other techniques such as quanti-tative buffy coat (QBC) and polymerase chain

    reaction (PCR) need expensive equipment and material and have not been adapted nor are feasible for the malaria field works in the endemic areas. Antibody detection by serologic tests usually shows prior exposure rather than current infection,3 particularly in the residents of malaria endemic areas.

    Therefore, the recent introduction of rapid diagnostic tests for malaria is of considerable interest. Such tests are based on antigen capture assay and application of the immunochromato-graphy (ICT) method for detection of Plasmodium specific antigen in a finger prick blood sample prepared from suspected malaria patients.3 The specific antigens for P. falciparum is a histidine-rich protein-II (Pf HRP-II) and Plasmodium lactate dehydrogenase (pLDH) for all human malaria parasites. These are soluble antigens released by the parasite and are detectable in the blood samples taken from parasitemic malaria patients. Detection of Pf HRP-II or pLDH antigens is performed by IgG monoclonal antibodies, which are prepared against these antigens.

    In the ICT method, monoclonal antibody is embedded on a clearly defined line (test line) present on a dipstick strip made of an absorbent laminated nitrocellulose/glass fiber. The Pf HRP-II or pLDH antigen is also deposited on the same strip in another line (control line) above the test line. A detection agent such as colloidal gold, which is conjugated with the antibody against the specific antigen, is used to indicate the control and positive test lines. Performance of the test takes about 10 minutes.1

    The main objective of this study is the evaluation of immunochromatographic test for P. falciparum "ICT Malaria Pf" in the detection of parasitemia in falciparum malaria infection and also in the in vivo drug susceptibility test.

    Materials and Methods

    Ninety-five dipstick test kits "ICT Malaria Pf" (ICT Diagnostics, Australia) containing: test cards, reagent A (lysing solution), capillary tubes coated with EDTA and product insert, describing test procedure, were received from the Reference Laboratory of the Iranian Ministry of Health and Medical Education for evaluation. The test kits were applied for diagnosis in suspected malaria patients as well as for the detection of parasitemia and follow up of chloroquine (CQ) treated falciparum malaria patients. These patients were under study of the in vivo extended drug susceptibility test4 which was performed in the Malaria Research Laboratory in Bandar Abbas Training and Health Research Center, Hormozgan Province, southeast Iran In these regions P. vivax and P. falciparum are prevalent and P. falciparum is more or less resistant to CQ.5

    The ICT Malaria Pf test was performed according to the test procedure described in the product insert briefly as follows:

    The test card was opened and the blood sample, taken by the capillary tube from the patient’s pricked finger, was put on the purple area of the sample pad of the dipstick strip located in the right hand side of the card. Once the purple pad was saturated with blood sample, one drop of reagent A was poured immediately above the purple pad (two drops below the blood spot and four drops on to a cleaning pad) located on the top of the left side of the card. After running up the lysed blood up to a limit line on the strip, the card was closed. The result could then be read through a viewing window 3-5 minutes after the color of blood has almost cleared.

    The test was considered positive when two lines were visible and negative when only a control line was observed. In case that no line or only the test line appeared, the result was considered to be invalid.

    The results of all ICT Malaria Pf tests were compared and evaluated with the results of the ME and parasite count against white blood cells (WBC) in the stained thick blood smears stained with Giemsa.

    Results

    From 52 suspected malaria patients, 31 had some blood forms of P. falciparum. They consisted of young trophozoites (TF), trophozoites and gametocytes (TGF), trophozoites with a few schizonts (TSchF), and only gametocytes (GF) in 17, 10, 2 and 2 cases, respectively. In 29 cases that asexual form of P. falciparum were detected in ME, the ICT was also positive. In the two cases that only gametocytes were observed, ICT was negative. In 8 cases that blood forms of P. vivax were detected and in 13 cases in which no parasite was found ICT was also negative (Table 1).

    Nineteen patients with falciparum malaria, who were treated with chloroquine (25mg/kg over 3 days), were followed up in the extended in vivo drug susceptibility test. Their blood samples were examined by ME and parasite count as well as by rapid ICT in the days 3, 7 and 14 after start of chloroquine administration (day 0).

    On day 3, in 9 patients, among 19 cases, that the parasite was observed in the forms of TF (7 cases), TGF (1 case) and GF (1 case), the ICT was also positive. In 2 cases the parasite was not detected against 2000 WBC in ME but the result of ICT was positive. In 2 cases with invalid ICT in which results the control line did not appear, no parasite was detected. In the remaining 6 cases with negative parasitological examinations, the ICT was also negative.

    On day 7, from the total 19 patients, 3 cases in whom a few TF were observed in ME, had positive ICT. In these patients the parasites were considered as CQ-resistant at RII level. Therefore, they were treated with sulfadoxine-pyrimethamine (SDX-PYR) and quinine (QNN). The ICT was positive in 10 cases in which no parasite was detected against 2000 - 4000 WBC in ME. In 6 other cases both ME and ICT were negative.

    On day 14 from the above 10 ICT-positive cases in which no parasite was detected in ME on day7, recrudescence of the parasite (TF) and clinical symptoms were observed in 5 patients. In one patient who did not have any clinical symptoms a few TF were detected in ME and the ICT was also positive. The strains of the parasite in these 6 patients were CQ-resistant at RI level and they were treated with SDX-PYR and QNN. In the other 4 cases, in which no parasite was found in ME and had positive ICT on day 7, both ME and ICT were negative on day 14. (Table 2). Totally, from 19 falciparum malaria patients studied, in 9 cases the parasite was chloroquine-resistant (3 at RII and 6 at RI levels) and in the other 10 cases the parasite was most probably, chloroquine-sensitive.

    Discussion

    Evaluation of the rapid test "ICT Malaria Pf" in the diagnosis of falciparum malaria infection in comparison to microscopic examination, showed that the test is 100% specific. But as it does not detect the mature gametocytes of P. falciparum3 the sensitivity of the test appears to be around 93%. On day 3 of the in-vivo test, ICT was positive in one patient in which only gametocytes were observed. In this case, most probably the asexual forms of the parasite were so scanty that they could not be detected in ME against 2000 WBCs. In comparison to ME such minor low sensitivity of ICT does not decrease the validity of such rapid test where its main application is for the detection of parasitemia in falciparum clinical infection.

    Application of ICT in the follow up of the treated patients showed that ICT is more sensitive than ME in the detection of low asexual parasitemia. On day 7, ICT was positive in 10 patients in which no parasite was detected in their blood samples against 2000 or even 4000 WBC in ME. From these ICT-positive cases seen on day 7, recrudescence of P. falciparum occurred in 6 patients up to day 14. But in the 6 parasitological and ICT-negative cases no parasites were found on day 7, or on day 14. This finding shows that ICT has predictive value in detecting low drug-resistant cases at RI level in some patients on day 7. However, in 4 cases that ICT was positive on day 7, it turned negative on day 14. This shows that antigenemia in some cases of P. falciparum as it has been reported before may persist in the blood of treated patients for 1 to 2 weeks after chemotherapy.3 Although the application of rapid ICT for assistance in the assessment of in vivo susceptibility needs further study with more samples, this preliminary finding shows that ICT could be used along with ME in the extended in vivo test on days 7 and 14 instead of the time consuming parasite count.

    At present for the diagnosis of malaria in endemic areas, the dipstick kits are rather expensive3 as compared to the microscopic examination. Nevertheless, it is very helpful for the remote areas as well as for clinics and health centers where the necessary facilities for ME are not accessible for the prompt diagnosis of falciparum infection, particularly in patients with severe and complicated malaria.

    References

    1. World Health Organization. A rapid dipstick antigen capture assay for the diagnosis of falciparum malaria. Bull Wld Hlth Org. 1996; 74: 47-54.
    2. World Health Organization. A global strategy for malaria control. WHO ; 1997.
    3. World Health Organization. Malaria diagnosis: new perspectives. WHO:CDS; 2000.14
    4. Payne D. Practical aspects of the in vivo testing for sensitivity of human Plasmodium spp to anti- malarials. WHO:MAP;1982.
    5. Edrissian GH, Nateghpour M, Afshar A, et al. Monitoring the response of P.falciparum and P.vivax to antimalarial drugs in the malarious areas in southeast Iran. Arch Irn Med. 1999; 2: 67-70.

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