Development of Monoclonal Antibodies That Target 1-Cys Peroxiredoxin and Differentiate <i>Plasmodium falciparum</i> from <i>P. vivax</i> and <i>P. knowlesi</i>

Prompt and accurate diagnosis of malarial patients is a crucial factor in controlling the morbidity and mortality of the disease. Effective treatment decisions require a correct diagnosis among mixed-species malarial patients. Differential diagnosis is particularly important in cases of <i>Plasmodium vivax</i>, a species that shares endemicity with <i>P. falciparum</i> in most endemic areas. Moreover, it is difficult to identify <i>P. knowlesi</i> on the basis of morphology alone, and rapid diagnostic tests are still not available for this malaria species. Therefore, the development of diagnostic tests applicable to the field is urgently needed. 1-Cys peroxiredoxin (1-Cys-Prx) in <i>P. falciparum</i> is abundantly expressed in the mature asexual stages, making it a promising candidate as a diagnostic antigen. In this study, we produced five monoclonal antibodies (mAbs) against <i>P. falciparum</i> 1-Cys-Prx (Pf1-Cys-Prx) by immunizing BALB/c mice with recombinant Pf1-Cys-Prx and subsequent hybridoma production. Cross reactivity of established mAbs with the orthologous molecule of Pf1-Cys-Prx in <i>P. vivax</i> (Pv1-Cys-Prx) and <i>P. knowlesi</i> (Pk1-Cys-Prx) was examined. Western blot analyses showed that three mAbs reacted with Pv1-Cys-Prx and Pk1-Cys-Prx but two mAbs did not. These results indicate that the two mAbs were effective in differentiating <i>P. falciparum</i> from <i>P. vivax</i> and <i>P. knowlesi</i> and could be used in differential diagnosis as well as comparative molecular studies of human <i>Plasmodium</i> species.

Characterization of the Population Demographics and the MSP-1 Block 2 Allele Gene Frequencies of <i>P. falciparum</i> Infected Individuals in Davao, Philippines

<i>Plasmodium falciparum</i> is one of the causative agents of malaria in humans. This parasite causes the most severe forms of the disease. In order to combat the disease, it is important to have knowledge about the parasite and its interaction with its host. In this study, we profiled 74 patients admitted to hospital in Tagum, Davao, Philippines who were confirmed to be infected with <i>P. falciparum</i>. We correlated the age, sex and parasite load with malaria severity and show that among these, only sex is correlated with disease severity in this population. In addition, we profiled the MSP-1 block 2 allele distribution in the population and found that the most abundant allele form was K1, followed by MAD20. The RO33 allele form was the rarest allele in this population.

Evaluation of blood preservation methods in the performance of the WHO in vitro micro-test for Plasmodium falciparum in the field

In vitro drug susceptibility testing of <I>Plasmodium falciparum</I> must be conducted immediately after collecting a sample of the patient‘s blood; otherwise the parasites may weaken and the culture fail. Collecting blood samples from individuals in areas far from the field station or clinic where in vitro testing is conducted requires a reliable method of sample preservation during transportation. We examined and compared three different methods used to preserve blood samples in endemic areas in the Philippines. The three methods are as follows: the on-site method (test is conducted soon after blood sampling), flask culture method (sample is taken to the laboratory in a culture flask with medium) and EDTA tube method (sample is taken to the laboratory in a blood collection tube). The WHO <I>in vitro</I> micro-test for susceptibility of <I>P. falciparum</I> to chloroquine was performed using an AnaeroPack® system and a portable thermostat incubator. Evaluation of the three methods was based on schizont maturation, ease of handling, and risk of contamination during the test. The on-site and flask culture methods, but not the EDTA tube method, were effective for keeping the parasites viable. Furthermore, schizont maturation appeared better with the flask method than with the on-site method, especially in the control wells (drug-free wells). In addition, it was easier to perform the flask method than the on-site method. No contamination was observed using any of the methods. The results of the study suggest that the flask culture method is the most effective and useful way to preserve blood samples for the in vitro test and, moreover, that it aids in providing detailed field evidence of drug-resistant malaria.

A PILOT FIELD TRIAL OF AN IN VITRO DRUG SUSCEPTIBILITY TEST USING THE ANAEROPACK MALARIA CULTURE SYSTEM ON THE THAI-MYANMAR BORDER

The AnaeroPack<SUP>®</SUP> malaria culture system with a portable thermostat incubator was evaluated in a field laboratory on the Thai-Myanmar border conducting <I>in vitro</I> drug susceptibility tests on blood samples from 5 Karen children infected with <I>P. falciparum</I>. Only one isolate was susceptible to chloroquine; the others were highly resistant. The IC<SUB>50</SUB> value of an isolate was only resistant to mefloquine, whereas the values of the 3 patients who presumably showed recrudescence were slightly elevated in the susceptible ranges. These results suggested that chloroquine should no longer be used for <I>P. falciparum</I> malaria in this geographic area, and that mefloquine should be carefully monitored for its <I>in vivo</I> effectiveness. In this study, the AnaeroPack<SUP>®</SUP> malaria culture system with portable thermostatic incubator is a powerful and useful mobile tool, which aids in providing detailed evidence-based distribution data concerning of drug resistant malaria in the field.