Indian Pediatrics 2001; 38: 650-654
Rapid Manual Test for Falciparum Malaria
From the Departments of Pediatrics and Phar-
macology and Division of Biostatistics, M.L.B. Medical College,
Jhansi (UP), India.
Malaria continues to be a major killer of mankind, especially in developing countries. According to the World Health Organization (WHO) more than 40% of the world’s population are exposed to the risk of malaria(1). Out of these, 270 million suffer from malaria and more than one million die of the disease every year. A key feature of WHOs new malaria control strategy is rapid diagnosis of malaria even at village level(2).
HRP-II antigen detection test is a rapid manual species-specific test for P. falciparum malaria. The test utilizes the detection of P. falciparum antigen based on the principle of immunochromatography. It is an antigen capture test(3), detecting trophozoite derived monoclonal antibody of soluble antigen (histidine rich protein II). It can be performed at primary health care level and gives result in fifteen minutes. In various studies(4-8), the sensitivity and specificity of HRP-II antigen detection test have been claimed to be very high. The aim of this study was to estimate the sensitivity and specificity of the test for falciparum malaria.
Subjects and Methods
The present study was conducted in the Department of Pediatrics, M.L.B. Medical College and Hospital, Jhansi (U.P.) on 192 children. Children upto the age of 12 years of either sex who attended the Outpatient Department or were admitted in Emergency and Pediatric wards as clinically suspected cases of malaria during Jan ’99 to Dec ’99, were included in the study.
Detailed general as well as systemic examinations were done for each case. Routine hematological profile was done in all the cases. Other causes of altered sensorium or convulsions were ruled out on the basis of investigations like electroencephalogram, X-ray chest, CSF, etc. Widal test and liver function tests were also done whenever required.
Peripheral blood smear examination for malarial parasite was done in all the cases. Thick and thin blood smears were made by using the edge of slide and another with the corner of slide. Slides were stained with Giemsa stain and were examined under light microscope by a single observer. Thick smears were examined for presence of parasite, while thin smears were evaluated for species of parasite. Atleast 100 high power fields were scanned in each slide. Results of thick and thin smears were recorded whether they were positive or negative for malaria for each child.
HRP-II Antigen Detection Test
Paracheck strips of Orchid Company were used in the study. Strip consists of a flattened fibre wick (3 cm long and 0.5 cm wide). A monoclonal antibody, raised against a portion of histidine rich protien II was applied in a line across the wick 7 mm away from the base and 15 mm from base; HRP-II antigen was applied to serve as control.
Paracheck strip was brought to room temperature and finger prick blood was delivered to the sampled area. Two-three drops of clearing buffer was taken in a glass tube and strip was dipped upto arrow on the dipstick. After 15 minutes, the reading was noted. It was taken as negative if only one pink color band appeared on dip stick and as positive for falciparum, if two distinct color bands appeared.
Post Treatment Sensitivity of HRP-II Antigen Detection Test
To evaluate post antimalarial therapy sensitivity of HRP-II test for P. falciparum on 4th and 10th day of treatment, only cases positive for P. falciparum on the day of admission were included. Information was recorded from cases who were receiving antimalarial drugs prior to hospital admission. On 4th and 10th day of antimalarial therapy, thick and thin smears and HRP-II test for P. falciparum were performed. Results of these tests were noted and findings compared.
On the basis of results of thick and thin blood smears, two groups were made.
One hundred and sixty children who were either negative for malaria or positive for species other than Plasmodium falciparum, constituted this group. There were 40 children positive for P. vivax in this group.
Out of 32 children in Falciparum group, HRP-II test was positive in 26, (sensitivity was 81.25% and specificity was 78.75%). To evaluate species specificity of HRP-II test, only those 40 children who were positive for P. vivax on thick-thin blood smear, were considered. HRP-II test was positive in four children (considered false positive) and negative in 36 children (considered true negative). Thus, the species specificity of HRP-II test for P. falciparum was 90%. Out of 160 children who were negative for P. falciparum on thick-thin blood smear examination, HRP-II test was positive in 34 children. Thus, false positivity of HRP-II test for P. falciparum was 21.25%. We also observed false negativity in 18.7% children in respect of blood smear.
Out of 60 children who were HRP-II test positive for P. falciparum on the day of admission and were on antimalarial therapy, 8 cases of cerebral malaria died within the first four days due to development of multiple complications like gastro-intestinal bleeding, hepatic coma, acute renal failure and shock. In the remaining 52, HRP-II test was positive in 36 (69.2%) and smear was positive in 6 (11.5%) on the fourth day. Out of these, only 12 children were available for HRP-II test on 10th day. Consideration of the disease to be ordinary by the community, dramatic response to the treatment and almost complete recovery within 3-4 days, were the main reasons for the poor follow up. In them, only 8 (66.7%) were found to be positive for HRP-II test and 2 (16.7%) for smear test (positive for gametes).
Twenty six children in this study were initially positive for falciparum by both the methods. Out of these, 19 were available on fourth day. In them, HRP-II test was positive in 13 whereas smear was positive in 6 children.
Solid phase inhibition radioimmunoassay and Elisa have been applied to demonstrate parasite antigen in solubilised erythrocytes. However, such tests require laboratory support facilities. HRP-II test can be per-formed at bed side and provides results within fifteen minutes(9).
The sensitivity and specificity of HRP-II test for P. falciparum is compared with other studies in Table I. The reason for relatively lower specificity in our study could be due to comparatively higher number of (21.25%) false positive HRP-II cases. Antimalarial drugs given prior to investigations decrease chances of positivity of blood smears in patients suffering from malaria. In our study, 25% children who were positive for HRP-II test had history suggestive of use of antimalarials along with other treatment for fever, probably affecting parasitemia but not the antigenemia. Since HRP-II test detects antigen, chances for positivity could be more than that of blood smear (gold standard considered for diagnosis of malaria in our study). In fact, the possibility of false negative blood smears in such children might be responsible for a comparatively larger sub group, labelled as false positive by us. Thus, HRP-II test has good potential to detect partially treated cases of malaria. Moreover, in P.vivax, P. ovale and P. malariae infec-tions, the entire asexual cycle of the parasite occurs in peripheral blood and the total number of parasites in the body can be estimated by examination of peripheral smears by light microscopy. However, in P. falciparum, the total burden of parasities can not be estimated since they disappear from the peripheral blood after 24-26 hrs of asexual development as a result of adherence to infected erythrocytes to the endothelium of venule and capillaries in the vital organs. Thus, if peripheral smear is examined after this stage, it may not detect parasite but the HRP-II test is not likely to miss the diagnosis. This could be another reason for discrepancy in both the tests.
In our study, the sensitivity of HRP-II test was 81.25% which was less than that of other studies (Table I). The sensitivity in these reports also varied between 88.9% to 98%. Human error in species identification during smear examination may be responsible for such variation, especially when old falci-parum (presence of gametes) and fresh vivax infections have occured, the temptation could be to report as falciparum, which is a more dreadful disease. HRP-II test will not detect falciparum gametes. Moreover, in partially treated or partially responsive patients, the extent of antigenemia might not be just enough to be detected by HRP-II test, being non quantitative in nature.
Specificity of HRP-II test by species in our study was 90%. Shiff et al.(6,7), in their studies showed species specificity to be 100%. To evaluate the post antimalarial therapy sensitivity of HRP-II test for P. falciparum only those cases were included who were positive by HRP-II test for P. falciparum. HRP-II test showed positivity of 69.2% on 4th day and 66.6% on 10th day. Our results are almost similar to those of other studies done previously(6,7,10). Positive readings by the HRP-II test within 10 days after treatment may occur in cured cases and does not necessarily mean treatment failure, because positivity following treatment is due to persistent parasite antigen rather than parasitemia. Thus, the HRP-II test is not only valuable for confirming malaria diagnosis on clinical cases, but has potential to detect recrudescent infections within two weeks of treatment. It would, therefore be seen that persistent P. falciparum HRP-II antigen, following treatment with chloroquine/quinine generally clears the infection by 10th day, beyond which the test may indicate a recrudescence.
HRP-II test has potential application in detection of chloroquine/quinine resistant falciparum malaria infections, since reinfec-tion with sensitive strains is unlikely upto 14th day after treatment(10). But it is not useful to assess the response of antimalarial therapy as the test remains positive for two weeks, whereas disappearance of parasitemia after a successful treatment can be demonstrated by blood smear test. It also fails to detect gametocytes in the blood. It has a dis-advantage in diagnosing partial response of treatment because the extent of antigenemia may not be depicted by the test results unless the test is quantitative.
In conclusion, though light microscopy is the mainstay of malarial diagnosis, it requires trained personnel and its reliability is questionable in partially treated cases of malaria. HRP-II test shows great promise for rapid and effective diagnosis of P. falciparum infection. The test is simple and can be easily performed even at clinics and village health centres. It requires minimal training, no special equipment is needed and the sensi-tivity and specificity of HRP-II test for P. falciparum are comparable to peripheral smear examination.
Contributors: AK was responsible for the concept and design of the study and drafting the manuscript; he will act as the guarantor for the study. SG collected and analyzed data. SK interpreted the data and revised the manuscript. BLV analyzed the data and interpreted the results.
Table I Comparison of Sensitivity and Specificity