|Year : 2000 | Volume
| Issue : 6 | Page : 227--232
Diagnostic accuracy of antigen capture dip - stick assay in falciparum malarial infection
HT Balakrishna1, Prema Saldanha2, Urmila Khadilkar2,
1 District Malaria Officer, District Laboratory, Hassan District, Karnataka, India
2 Department of Pathology, Kasturba Medical College, Mangalore- 575001, India
Department of Pathology, Kasturba Medical College, Mangalore- 575001
|How to cite this article:|
Balakrishna H T, Saldanha P, Khadilkar U. Diagnostic accuracy of antigen capture dip - stick assay in falciparum malarial infection.Indian J Med Sci 2000;54:227-232
|How to cite this URL:|
Balakrishna H T, Saldanha P, Khadilkar U. Diagnostic accuracy of antigen capture dip - stick assay in falciparum malarial infection. Indian J Med Sci [serial online] 2000 [cited 2015 Jul 3 ];54:227-232
Available from: http://www.indianjmedsci.org/text.asp?2000/54/6/227/43289
The resurgence of malaria in our country and in particular in Mangalore City made it essential for us to embark upon a better and quicker diagnostic method for malaria detection. In the bygone years, the conventional blood smears were the sole determinants of the presence of malarial infection. In course of time, the emergence of the Quantitative Buffy Coat [QBG] technique refind the diagnostic acumen. Yet there were limitations regarding the accurate identification of the species and the parasite stage [particularly in Plasmodium falciparum (Pf) infection], by the QBC method. This forced researchers to look for newer techniques for malaria detection and led to the evolution of the Dip-stick antigen capture assay [Parasight-TMF], which relies on the immunogenicity of the parasite, Pf. In the present study, we have made an attempt to compare the diagnostic accuracy of this newer method with that of QBC technique and the erstwhile blood smear.
Material and Methods
Blood samples were collected from 250 individuals with or without symptoms of malaria, in areas endemic for Pf infection in the Hassan District of Karnataka State, as well as from symptomatic individuals admitted at our hospital and other hospitals in Mangalore. In the symptomatic group, spiky fever associated with intractable headache and vomiting were the frequent complaints. The venous blood samples were collected in EDTA, placed in ice and transported to the , laboratory within 6 to 8 hours of collection. Thick and thin blood films were made and stained with Leishman's stain. The smears were examined for asexual forms of Pf. The negative slides were reread by counting upto 2000 leucocytes before classifying the sample as negative. The parasitaemia was confirmed by the QBC rapid fluorescent technique, which could be done in only 118 samples. In this method the parasite density was estimated by counting the asexual forms against 200 leucocytes [parasites/µl parasite count x leucocytes counted]. The species identification was confirmed independently by another microscopist.
The performance of the Parasight-TMF [Becton-Dickinson Company] was determined by correlation with the standard thick and thin blood smear method and the QBC technique. Parasight-TMF is a test incorporating a dual antibody immuno-assay against the Pf HRP-II antigen. Mouse monoclonal antibodies directed against the synthetic peptide [AHH (AHHAAD) 2 ]  from Pf HRPAI are immobilized on a test strip. Pf antigen in lysed fresh whole blood binds to the antibody as the blood absorbs into the test strip. The antigen immobilization is indicated as a "dash" just above the mouse atnibody line.
Pf HRP-II antigen-detector reagent contains rabbit polyclonal antibodies directed against HRP-II antigen, that are conjugated to liposomes containing pink dye. These antibodies bind to the antigens on the strip giving a control "dash". A solid pink line indicaes a positive test. A "dashed" pink control line and a white to light background will always be present if the test has been performed properly. The species specificity of the dip-stick essay was evaluated by testing with blood samples from cases having non-falciparum malaria [Plasmodium vivax (Pv)] and mixed infection [Pf and Pv]. The cross-reactivity was assessed in non-plasmodium infections like Wuchereria bacrofti.
A total of 250 samples were collected for analysis. 104 samples were from Hassan District, 93 patients being symptomatic and 14 asymptomatic. 146 samples were collected from patients with symptoms of Pf infection in Mangalore. Out of the 250 samples, 152 were positive by the dip-stick assay, 94 were negative and 4 were uninterpretable. The 152 samples, studied by blood film had asexual forms of Pf in 148, while 4 were smear negative. Among the 94 samples which were dip-stick negative, 93 were also smear negative and only one showed occasional parasites. The 4 samples uninterpretable by the dipstick test were found to have negative smears [Table 1]. The 118 samples tested positive by dipstick which were subjected to QBC analysis were all positive. The end-point sensitivity of the dip-stick [Parasight-TMF] test as compared to blood film examination at various levels of parasitaemia is tabulated in [Table 2]. The species specificity and cross-reactivity are illustrated in [Table 3]. The level of parasitaemia in the symptomatic and asymptomatic groups, and the mean standard deviations are given in [Table 4]. Of the 34 patients followed, 4 cases became dip-stick and smear negative on the same day, 10 remained dip-stick positive for 1 to 2 days, and 11 off 7 to 15 days after the smear was negative. No case became dip-stick negative before the blood film.
Detection of Pf antigen by radioimmuno-assay using a cross reacting antigen was first attempted by Avidor, but proved to be difficult and expensive.  It has been found that Plasmodium falciparum infected erythrocytes synthesize 3 histidine - rich proteins (HRP I or the knob - associated HRP, HRP II, and HRP III or SHARP.  HRP II is of highest abundance and its synthesis begins with the ring forms and continues through the trophozoie stage .  HRP II is a water-soluble protein. It has been found in all natural isolates of Pf tested, and has been detected in plasma and whole blood.  It has also been found in urine.  The current demonstration of Pf HRP 11 in plasma, plus the fact that it is a structurally well-characterized molecule present in all natural isolates of Pf tested, made Pf HRP II of interest for its potential effects on the host immune system and as an antigen for specific diagnosis of malaria. ,,,, The Parasight-TMF is a quantitative/semi-quantitative method which depends on the amount of Pf antigen present. An evaluation of this dip-stick technique (in which a dual antibody incorporated into a paper strip, captures the HRP II antigen of Pr) was undertaken in 250 individuals, 14 of whom were asymptomatic in an area endemic for falciparum malaria, and the remaining 236 were febrile. Out of this total of 250 samples tested, 152 were positive, 94 negative and 4 uninterpretable by the dip-stick assay [Table 1]. Only 118 samples were simultaneously subjected to QBC testing by fluorescent staining and all were identified positive. 113 of the 118 QBC positive samples were febrile and 5 were asymptomatic. The level of parasitaemia in the asympiomatic group was <103µl with a mean standard deviation (MSD) of 1.857 ± 4.40, and <10/µl with a MSD of 15.147 ± 44.466 in the symptomatic group. The difference in the mean of the two groups was found to be highly significant by the 't' test [Table 4]. A thick and thin smear examination of the 152 dipstick positive cases showed a sensitivity of 99.3%, specificity of 95.87% , a positive predictive value of 97.36% and a negative predictive value of 98.95% [Table 1].
Since most individuals with symptomatic Pf infections have <60 padasites/µl, the dip-stick assay will be of particular use in the rapid diagnosis of febrile patients and in epidemiologic field studies. Furthermore, because inexperienced microscopists often have difficulty in detecting <60 parasites/µl, assessment of the comparative sensitivity of blood film and dipstick assay among such technicians may indicate that the dip-stick has greater sensitivity than the blood smear. Among the 148 individuals who were positive by both drip-stick and blood film, 34 were followed long enough to have negative blood films. Among these, in 4 cases both tests became negative on the same day. 11 had a positive dip-stick 7 to 15 days after the blood film became negative, 10 remained dip-stick positive for only 1 to 2 days after the blood smear was negative, but no case became dip-stick negative before the blood smear. These data indicated that persistence of circulating antigen after the treatment might have accounted for the discrepancies between the dip-stick test and blood smear results. In the 4 cases which were positive by dip-stick and negative by blood film, a positive smear was reported at least once within the previous 20 days in 3 of the cases. The very rare occurence of a false negative Parasight-TMF test with a positive blood film could not be explained. Parasight-TMF is a simple test which requires no special laboratory equipment, is speedy, with the test procedure spanning only 10 minutes and is a specific method of detecting Pf infections obviating the need of a trained microscopist.
Parasight TMF antigen capture assay is comparable to the rapid OBC fluorescent technique in detecting malarial infection, even in low parasitaemia. A comparative analysis of the Parasight TMF assay with the conventional, blood smear examination showed a sensitivity of 99.3% specificity of 95.87%, a positive predictive value of 97.36% and a negative predictive value of 98.9%. An apparent false positive result may be expected with the Parasight TMF because the assay detects the antigenaemia even after elimination of viable parasites from the blood stream. Hence the decline in antigenaemia after radical curative therapy could be a useful parameter to monitor the Plasmodium falciparum malarial infection. The Parasight TMF assay is useful in detecting even subclinical falciparum malarial infections in asymptomatic individuals with a mean parasite count of <10/p1. The very rare occurrence of a false negative Parasight TMF test with a positive blood film could not be explained. The Parasight TMF test is specific for P. falciparurn as there was no cross-reactivity in the antigen-capture of P. vivax or Microfilarial infections. Parasight TMF test is a simple test which requires no special laboratory equipment, it is speedy with the test procedure spanning only 10 minutes, and is a specific method of detecting P. falciparum infections, at the Primary Health Centres, obviating the need of a trained microscopist.
The authosr are grateful to Dr. CV Raghuveer, Professor of Pathology for the critical comments and to Dr. KR Shetty, former Director, University Medical Centre, for providing the financial support in procuring the Parasight-TMF test kit for evaluation.
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