S. A. Khan  ( Department of Pathology, Army Medical College and Military Hospital, Rawalpindi. )
M. Anwar  ( Department of Pathology, Army Medical College and Military Hospital, Rawalpindi. )
S. Hussain  ( Department of Pathology, Army Medical College and Military Hospital, Rawalpindi. )
A. H. Qureshi  ( Department of Pathology, Army Medical College and Military Hospital, Rawalpindi. )
M. Ahmad  ( Department of Pathology, Army Medical College and Military Hospital, Rawalpindi. )
S. Afzal  ( Department of Pathology, Army Medical College and Military Hospital, Rawalpindi. )

Malaria remains an important parasitic disease worldwide. Three to five hundred million people contract the disease annually. It is estimated to kill between 1.5-3.5 million people each year.1 Prompt and accurate diagnosis is the key to effective disease management. Therefore it is one of the main interventions of the global malaria control strategy.2

Several approaches are available for the diagnosis of Malaria. Clinical diagnosis, still widely used could be only 50 % accurate.3 Examination of thin and thick blood film under the light microscope is considered a gold standard in the diagnosis of malaria. It is a sensitive, informative, inexpensive but labor-intensive method, requiring at least 60 minutes from specimen collection to result.4 Fluorescent microscopy is sensitive especially quantitative buffy coat examination. This method can screen quickly many samples but requires special equipment and training.5,6 Malarial pigment can be screened by dark-field microscopy but sensitivity is low.7 Polymerase chain reaction based molecular assays are the most sensitive but it cannot be employed for routine purposes.8 Latest automated haematology analyzers can flag suspicious samples by detecting malarial pigment in the white blood cells but still requires confirmation by another method.9

Development of malarial antigen based rapid diagnostic tests (Rdts) for detection of malaria in the past few years might offer a valid alternative to microscopy.7 These tests are based on the detection of antigens derived from malaria parasites in lysed blood, using immunochromatographic methods. Most frequently they employ a dipstick or test strip bearing monoclonal antibodies directed against the parasite antigens. These tests can be preformed in 15-20 minutes.10,11 Several commercial kits are available detecting two different malarial antigens. Histidine-rich protein2 (HRP-2) is a water soluble protein detected by ICT Malaria and parasite-F kits. These detect plasmodium falciparum only and the test is positive for some days even after successful treatment.12,13 Another dipstick assay (Optimal) detects plasmodium specific lactate dehydrogenase (pLDH). It can separate plasmodium species by detecting antigenic differences between various p-LDH isoenzymes. The test quickly becomes negative with treatment and promises to be quite sensitive and specific.14

We evaluated the performance of Optimal by comparing its sensitivity and specificity with light microscopy in detecting malarial parasite in patients with presumptive clinical diagnosis of malaria.

Two hundred and fifteen patients reporting sick from August 2002 to October 2002 in Military Hospital Rawalpindi with symptoms suggestive of malaria were included in the study. The clinical presumption of malaria was based on febrile illness of short duration associated with chills, rigors and body aches. Patients of all ages were included in the study. A detailed history and clinical examination was undertaken in all these subjects. Patients having any other obvious reason for fever and those who had taken antimalarials were excluded from the study. Three ml venous blood was collected in ethlylenediamine tetra acetic-acid anticoagulated bottles from each patient. Thin and thick blood smears were prepared, stained with Leishman's stain and examined for malarial parasite by light microscopy using 100 × oil immersion. Parasite number was counted per 200 white blood cells in P. falciparum positive thick smears. Parasite density was expressed as number of parasite / ul. A blood smear (thin and thick) was considered negative if no parasite was seen after 10 minutes of search or examination of 100 high power fields of microscope.1,9

Same blood samples were also tested by Optimal dipstick method according to manufacture's (Flow inc. Portland. OR, USA) instructions. The immunochromatographic test detects the presence of pLDH antigen in lysed whole blood. pLDH is released from live malarial parasites and differentiation of plasmodium species is based on antigen differences between its isoforms. Aside from a control antibody reaction zone at the top of the test strip, the optimal dipstick contains two test lines or reaction zones. The first line encountered by the sample comprises of an antibody that is specific for P. falciparum pLDH. The second test line is composed of a pan specific pLDH monoclonal antibody that recognizes all other plasmodium species (P.vivax, P. malaraie and P. ovale).

Two drops of reagent A (30 ul of colloid/ buffer solution) were added to the conjugate well and four drops of reagent B 8 ul of clearing solution) were added into the wash well provided on a configured well plate. Ten microlitres of blood was added to the conjugate well and mixed gently. Dipstick was placed vertically into the conjugate well and allowed to stand for 10 minutes. The dipstick was then transferred to wash well and left there until the bands became clearly visible within 5-10 minutes. The interpretation of results was performed immediately after completion of the clearing step as follows:

1. Positive - P. falciparum; one control band and two test bands.
2. Positive - P. vivax and other plasmodium species; one control band and one test band.
3. Negative - One control band at the top of the strip. Examples are shown in Figure.

positive, 58 were P. vivax and 35 P. falciparum. These results demonstrated that optimal has sensitivities of 95% and 94.5% for P. Vivax and P. falciparum respectively, when compared to conventional

microscopy. It has 100% specificity for both malarial species. Optimal dipstick method missed two cases of P. falciparum that were positive by microscopy and these were the cases who had a low parasitic density. The comparison of sensitivities of two methods in respect of P.falciparum density is shown in Table.

Two cases of mixed malarial infestation were observed on microscopy. In both these cases Optimal gave the result as P. falciparum. One case showed only gametocytes of P. falciparum and no ring forms were seen on

microscopy. Optimal showed the case positive for P. falciparum infestation. It required minimum sixty minutes to deal with one blood sample by microscopy whereas with Optimal method, the results were available within 15-20 minutes.

The study compared the results of a new rapid malarial diagnostic method (0ptimal) with traditional light microscopy. It was found that both the methods yielded comparable results. Optimal missed two cases of P. falciparum and three cases of P. vivax which were positive on microscopy. There could be two reasons for these false negative results. Firstly, 0ptimal is not sensitive below a parasitic index of 100 parasite/ul.15 It is also reflected in our results as all the P. falciparum cases which were missed by 0ptimal had a parasitic density below 100 parasite/ul. Secondly, 0ptimal detects pLDH which is produced by living parasites. It is possible that some patients might have already taken antimalarials earlier and had not disclosed it.

For clinical use the sensitivity of the Optimal as currently configured should be adequate for malarial diagnosis. Majority of patients of P. falciparum infestation (as in this study 58 out of 61) usually have parasitic index greater than 100 parasite/ul. This is also true for P. falciparum infestation in non-endemic areas and in non-immune patients. Microscopy can pick malarial parasites even if the density is as low as 10-50 parasites/ul.9,15 This sensitivity of microscopy is seen under study conditions where trained microscopists are scanning each blood smear for 10-20 minutes which does not seem practical in a busy hospital setting in routine. We are of the view that in routine hospital laboratory practice the sensitivity of Optimal should be comparable to microscopy or even more. Optimal does not have any subject bias whereas in microscopy the results are affected by the skill and workload of the microscopists.

Two cases of mixed malarial infestation were observed on microscopy. Optimal reported these cases as P. falciparum due to the configuration of the test. This inevitably missed the other species, but as P. falciparum is the species which causes the most majority of clinical complications, the test format is not disadvantageous. One case in this study showed only the gametocytes of P. falciparum in the blood smear and no ring forms were seen. Optimal reported it as P. falcipurum infestation as pLDH is secreted by gametocytes. These Optimal results can lead to unnecessary medication.

Several authors have previously reported trials of Optimal for malarial diagnosis from different countries with more or less similar results. Palmer et al16 in Honduras has reported a sensitivity of 94% and specificity of 100% for P. vivax and sensitivity of 88% and specificity of 99% for P. falciparum. From hospital of tropical diseases UK, Moody et al17 while conducting their study in sub-Saharan Africa, Asia and South America have reported a sensitivity of 95.3% and specificity of 100% for P. falciparum and sensitivity of 96% and a specificity of 100% for plasmodium vivax. John et al18 obtained a sensitivity of 94% for P. falciparum and 98.2% for P. vivax in a trial in Southern India. In Gambia, Hunte-Cooke et al19 reported a sensitivity of 91.3% and a specificity of 94% for P. falciparum with Optimal. A short study carried out at Combined Military Hospital Quetta by Ahmad et al20 has reported a sensitivity and specificity of 100% for both species. Our results are comparable in sensitivity and specificity from most of the above mentioned studies carried out in different parts of the world. Therefore the results of this study further substantiates that 0ptimal is an effective and sensitive tool in the diagnosis of

Microscopy is the gold standard for diagnosing malaria. Although it is time consuming, as one test requires 60 minutes, but it is economical and accurate if performed by a patient and skilled technologist. In comparison, the Optimal test is simple more objective, requires no equipment, but is more expensive. However, the cost factor becomes less significant due to the reduced morbidity and hospital admissions.

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