**7. Three new parasite ldh (pan-pLDH) tests for diagnosis of uncomplicated malaria**

Since Charles Laveran first visualized the malaria parasite in 1880; the mainstay of malaria diagnosis has been the microscopic examination of blood smear. It is the main economic, most preferred and reliable diagnosis using two types of blood films, which is amenable to the four species of malaria parasite. These two types of films are (a) the thin blood film which is similar to the usual blood films, which allows species to be identified because they can be visualized and the appearance of the parasites are much more distorted.(b)the thick film from which an experienced microscopist can detect parasite levels or parasitemia down to levels as low as 0.0000001% of red blood cells. Diagnosis of species can be difficult because, the early trophozoites ("ring form") of all four species look identical and never possible to diagnose the species on the basis of simple ring form. The success of the method above requires well trained staff, quality equipment and supervision. The scarcity of these facilities within malaria endemic areas becomes limiting. In sub-Saharan Africa and some other areas, clinicians often have to rely on clinical signs and symptoms for diagnosis and in some areas where increasing emphasis is laid on home based management , malaria diagnosis is often equated[11] with fever. It is to be noted that such presumptive treatment without laboratory confirmation could contribute to the development of drug resistance[11]. Today, an alternative method to the blood film diagnosis approach is the rapid diagnostic test (RDTs), recommended by WHO, where reliable microscopy is not reliable or available. Rapid diagnostic tests (RDTs) are antigen detection tests, which are simple to use and interpret, although the tests also use peripheral blood. The most commonly used RDTs, is the histidinerich protein 2 (HRP 2), produced by trophozoites and young gametocytes of plasmodium falciparum. HRP 2 test has been the most widely evaluated to date test, and has shown consistently high sensitivity. The limitation of this test is that RDTs detect P. falciparum only and can remain positive for several weeks after antimalarial treatment. Besides these, a study to assess the diagnostic capabilities of three parasite lactate dehydrogenase (pan- LDH)- ,Vistapan® ,Carestat™ and Parabank® were conducted in Uganda. Similarly, a histidine- rich protein 2 (HPR 2) test, Paracheck-Pf® and a Geimsa-stained blood film were performed with pfLDH tests for outpatients. A total of 460 patients were recruited for the exercise, 248 had positive blood films and 212 with negative blood films. *Plasmodium falciparum* was present in 95% of infections. Sensitivity of the tests above 90% was shown by two pLDH tests-Carestart (96.5%) and Vistapan (91.9%) and sensitivity above 90% by Parabank (94.3%) and Carestart (91.5%). The benefits of these tests when compared with the previous gold standard for laboratory confirmation of malaria diagnosis which is a peripheral blood film examined microscopically shows the high specificity and validity of the tests.

Functions of Dehydrogenases in Health and Disease 171

thus being cleared from the blood stream more rapidly after treatment, resulting in test becoming negative more quickly. There is no doubt that these characteristics suggest that pLDH tests could be used with more confidence for malaria diagnosis at the peripheral level. The development of several new pLDH monoclonal antibodies by Flow Inc. has enabled the production of a new generation of pLDH tests. These characteristics suggest that pLDH tests could be used with more confidence for malaria diagnosis at the peripheral level [14]. For confident diagnosis of malaria in routine outpatient department conditions, a sensitivity of more than 90% is crucial and this has been achieved by both Carestart and Vistapan. The pLDH tests have also demonstrated desirable qualities that could reduce the possibility of patients without malaria being given antimalarials, which may therefore reduce drug pressure, a major concern at a time when Artemesinin combination therapy (ACTs) are being introduced in Africa. The validation of these tests for malaria diagnosis involves many stages from the selection of site, enrolment of patients, sample size, study procedures, laboratory procedures, study outcomes, analysis and results. The validity of the tests after all these procedures must be rated from 90% and above. From the validity tests carried out by some researchers, Carestart had estimates for all validity parameters greater than 90%. Vistapan and Carestart were also sensitive as Paracheck Pf (p=0.14 and p=0.38) respectively. Parabank was less sensitive than all other tests (P<0.001 for each comparison). There was no significant difference between the three pLDH tests, but Parabank had a higher specificity compared with Paracheck Pf (P=0.02) for P. falciparum detection. In the study, the ages of patients were taken into consideration. Sensitivity decreased with older age for both Vistapan (97.4%) for the under fives versus 85.7%, P<0.011 and Parabank (95.4%) for the under fives versus 73.1%, P<0.001. Three tables were used to summarize the results of a study conducted in Mbarara Regional Hospital in Uganda, in a mesoendemic area of malaria transmission. These results are shown in tables 2, 3 and 4 respectively.

The first approach is to select a site, which should be a highly malarial infested zone.

Patients from the outpatient department were systematically screened for symptoms suspected to be malaria and referred to the research clinic. Inclusion criteria were a clinical suspicion of malaria; weight≥ 5kg; resident in Mbarara Municipality available for two weeks follow up period; and signed informed consent from the study subjects or their legal guardians. Exclusion criteria were signs of severe or complicated malaria [15b], signs of severe disease; and women with visible pregnancy or suspicion of pregnancy based on the

The required number of patients with positive blood film was calculated using an estimated sensitivity of the RDTs of 90%, an alpha error of 0.05 and a precision of 6%. This number (n-

**7.1. Materials and methods** 

**7.2. Enrolment for the study.** 

**7.3. Sample size** 

assessment of the last normal menstrual period.

An alternative diagnostic method to the rapid diagnostic test (RTD), recommended by WHO, where reliable microscopy is not available. RTDs are antigen detection tests, which are simple to use and interpret and also use peripheral blood. The most commonly used RTD detects histidine -rich protein 2 (HRP2), produced by trophozoites and young gametocytes of *Plasmodium falciparum*. HRP 2 tests have been the most widely evaluated to date and show high sensitivity. However, they are limited in that they detect P. falciparum only and can remain positive for several weeks after successful treatment[12,13].

The second type of RTD detects the malaria antigen parasite lactate dehydrogenase (pLDH), an enzyme produced in the glycolytic cycle of the asexual stage of all species of Plasmodium. Parasite lactate dehydrogenase( pLDH) are produced only by viable parasites, thus being cleared from the blood stream more rapidly after treatment, resulting in test becoming negative more quickly. There is no doubt that these characteristics suggest that pLDH tests could be used with more confidence for malaria diagnosis at the peripheral level. The development of several new pLDH monoclonal antibodies by Flow Inc. has enabled the production of a new generation of pLDH tests. These characteristics suggest that pLDH tests could be used with more confidence for malaria diagnosis at the peripheral level [14]. For confident diagnosis of malaria in routine outpatient department conditions, a sensitivity of more than 90% is crucial and this has been achieved by both Carestart and Vistapan. The pLDH tests have also demonstrated desirable qualities that could reduce the possibility of patients without malaria being given antimalarials, which may therefore reduce drug pressure, a major concern at a time when Artemesinin combination therapy (ACTs) are being introduced in Africa. The validation of these tests for malaria diagnosis involves many stages from the selection of site, enrolment of patients, sample size, study procedures, laboratory procedures, study outcomes, analysis and results. The validity of the tests after all these procedures must be rated from 90% and above. From the validity tests carried out by some researchers, Carestart had estimates for all validity parameters greater than 90%. Vistapan and Carestart were also sensitive as Paracheck Pf (p=0.14 and p=0.38) respectively. Parabank was less sensitive than all other tests (P<0.001 for each comparison). There was no significant difference between the three pLDH tests, but Parabank had a higher specificity compared with Paracheck Pf (P=0.02) for P. falciparum detection. In the study, the ages of patients were taken into consideration. Sensitivity decreased with older age for both Vistapan (97.4%) for the under fives versus 85.7%, P<0.011 and Parabank (95.4%) for the under fives versus 73.1%, P<0.001. Three tables were used to summarize the results of a study conducted in Mbarara Regional Hospital in Uganda, in a mesoendemic area of malaria transmission. These results are shown in tables 2, 3 and 4 respectively.
