**8. Comparison of two rapid field immunochromatographic tests to expert microscopy in malaria diagnosis.**

The vast majority of malarial tests adopted and used in the past depended on light microscopy and expertise in attaining results. Currently, the vast majority of malaria cases in the world are diagnosed by the century old standard of light microscopy and stained blood smears. Although the technique is sensitive and very inexpensive, there are several disadvantages. The disadvantages include practical issues such as electrical requirement, the need for experienced staff as well as difficulties in accurate species identification. Efforts have been made to develop malaria rapid diagnostic devices (MRDDs) to facilitate field diagnosis [18]. While the first generation tests diagnosed only Plasmodium falciparum, newer devices were designed to recognize both Plasmodium falciparum and P. falciparum specific antigen as well as Plasmodium genus specific antigen.

The ICT malaria Pf/P.v test is a rapid immunochromatographic assay for the detection of P. falciparum-specific Pf HRP 2 and a pan-malarial antigen, manufactures in test card form. The name is misleading as the pan malarial antigen may be produced by Plasmodium ovale, and Plasmodium malariae as well. The test has been shown to be specific and sensitive. Malaria P.f/P.v test has been shown to be 96% sensitive and 90% specific for P. falciparum and 75% sensitive and 95% specific for P. vivax [19,20].

The OptiMAL dipstick test detects two forms of plasmodium lactate dehydrogenase parasites pLDH1; one P. falciparum-specific and one common to the four plasmodium species which infect humans. Many researchers reported a 95% sensitivity and 100% specificity for P. falciparum, and a 96 % sensitivity and 100% specificity for P.vivax. Sensitivity for P. ovale and P.malariae were significantly lower (57% and 47%) respectively. Using a batch OptiMAL dipstick procedure in 1999, a large scale field evaluation showed the following result: 91% sensitivity and 95% specificity for P.falciparum and 83% sensitivity and 100 % specificity for P.vivax [21].

Methodology. The methodology involved the use of patients both symptomatic and asymptomatic , who are villagers referred to the study and informed consent obtained. For each patient, a finger prick was made and the following were obtained. Fifty microliters of blood in a pre-heparinized Eppendorf tube for dipstick assays and two thick/ thin smears, one for on –site microscopic diagnosis by Acridine orange(AO) technique (for immediate treatment purposes, and the other, for reference Geimsa-microscopist.OptiMAL reader and ICT reader, were all blinded to each other's diagnosis. Patients positive for malaria were treated. Thick and thin blood smears were prepared and stained with Geimsa according to standard procedures. To declare a sample negative, thick smears were read for 200 microscopic fields (1000 X) without finding a parasite.If found positive, the number of asexual malaria parasites were counted per 500 WBC separately for each species. If there are more than 250 parasites/500 WBC, parasites were counted on the corresponding thin film per 10,000RBC. Density calculations were based on approximations of7500 WBC/µl and 5X10 6 RBC/µl. ICT malaria P.f/P.v test kits were used as per manufacturers instructions. Ten microliters (10µl) of whol blood was transferred to a sample pad. A buffer reagent was added to induce cell lysis and allow PfHRP 2 and pan malarial antigens to bind to colloidal gold-labeled antibodies. Additional buffer caused the blood and immune complexes to migrate up the test strip and cross monoclonal (mAb) lines. Finally, more buffer was added to clear blood from the membrane and facilitate reading.

176 Dehydrogenases

The pLDH tests also demonstrated desirable qualities that could reduce the possibility of patients without malaria being treated or given antimalarial drugs, thereby reducing drug pressure and resistance, a major concern at a time when artemisinin combination therapy (ACTs) are being introduced throughout Africa. Their high specificity would reduce the number of patients with false positive results. Secondly, the great reduction in the number of tests remaining positive for a long time after treatment had been effected. This is reminiscent of the test carried out by HRP 2. Thirdly, the ability to detect both P. falciparum and P. vivax, would increase confidence on a negative test result. A variety of factors may contribute towards differing sensitivities of the test, such as patients age and level of parasitemia, which will vary according to endemic nature of P. falciparum in the locality. Lower test sensitivity may be related to low parasitemia in adults in an area of stable transmission. This may be a limitation of the tests; although, such patients are less at risk

**8. Comparison of two rapid field immunochromatographic tests to expert** 

The vast majority of malarial tests adopted and used in the past depended on light microscopy and expertise in attaining results. Currently, the vast majority of malaria cases in the world are diagnosed by the century old standard of light microscopy and stained blood smears. Although the technique is sensitive and very inexpensive, there are several disadvantages. The disadvantages include practical issues such as electrical requirement, the need for experienced staff as well as difficulties in accurate species identification. Efforts have been made to develop malaria rapid diagnostic devices (MRDDs) to facilitate field diagnosis [18]. While the first generation tests diagnosed only Plasmodium falciparum, newer devices were designed to recognize both Plasmodium falciparum and P. falciparum

The ICT malaria Pf/P.v test is a rapid immunochromatographic assay for the detection of P. falciparum-specific Pf HRP 2 and a pan-malarial antigen, manufactures in test card form. The name is misleading as the pan malarial antigen may be produced by Plasmodium ovale, and Plasmodium malariae as well. The test has been shown to be specific and sensitive. Malaria P.f/P.v test has been shown to be 96% sensitive and 90% specific for P. falciparum

The OptiMAL dipstick test detects two forms of plasmodium lactate dehydrogenase parasites pLDH1; one P. falciparum-specific and one common to the four plasmodium species which infect humans. Many researchers reported a 95% sensitivity and 100% specificity for P. falciparum, and a 96 % sensitivity and 100% specificity for P.vivax. Sensitivity for P. ovale and P.malariae were significantly lower (57% and 47%) respectively. Using a batch OptiMAL dipstick procedure in 1999, a large scale field evaluation showed the following result: 91% sensitivity and 95% specificity for P.falciparum and 83% sensitivity

from severe clinical episodes to perpetuate parasite transmission.

specific antigen as well as Plasmodium genus specific antigen.

and 75% sensitive and 95% specific for P. vivax [19,20].

and 100 % specificity for P.vivax [21].

**microscopy in malaria diagnosis.** 

Tests are counted as valid, if control lines are observed. They were counted P. falciparum positive, if Pf HRP 2 specific and pan-malarial antigen lines were visible or if only PfHRP 2 specific lines were seen only. If the control and pan-malarial antigen lines were observed, the sample was counted as positive for a malaria parasite other than P. falciparum. The test result was assigned a value of +0, if no line was seen; +1, if test line intensity was less than control line intensity; +2,if it was equal and +3, if it was greater in intensity. Other factors can exacerbate drug resistance. The second type of RDT detects the malarial antigen parasitelactate dehydrogenase (pLDH), an enzyme produced in the glycolytic pathway or cycle of the asexual stage of all species of plasmodium. Parasite lactate dehydrogenase is produced only by viable parasites, being cleared from the blood stream more quickly after treatment, resulting in the test becoming negative more quickly [22].
