DOI: 10.14457/tu.the.2015.1561
title: The roles of heme-oxygenase-1 and Prostaglandin D2 in pathogenesis and severity of severe falciparum malaria and genetic diversity of antimalarial drug resistance genes in Plasmodium falciparum and Plasmodium vivax

description: Malaria remains a major public health problem in the world.  Plasmodium falciparum and Plasmodium vivax are the two major malaria species responsible for most cases of malaria in Thailand.  Thorough understanding of disease pathogenesis, availability of rapid diagnosis, and efficient system for detection of antimalarial drug resistance are the key components for effective malaria control.  Through a series of research projects, the current dissertation proposal aimed to investigate: (i) molecular mechanisms of pathogenesis and severity of P. falciparum malaria; (ii) the application of a new ELISA-based test as an alternative malaria diagnostic tool; and (iii) the distribution and diversity of genetic markers of antimalarial drug resistance in           P. falciparum and P. vivax isolates from the endemic areas in Thailand, and in addition in the Philippines, Bangladesh and Nepal.The role of heme-oxygenase-1 (HO-1) in malaria pathogenesis and severity was investigated in vitro using the co-culture model of human brain microvascular endothelial cell (HBMEC) and ITG Plasmodium falciparum-infected red cells (iRBC).  The enzyme is involved in heme degradation process to release the nontoxic products iron, carbon monoxide, and biliverdin/bilirubin.  Following exposure to the HO-1 inducer CoPPIX at all concentrations, the HBMEC cells apoptosis occurred which could be prominently observed at 15 µM of 3 hours exposure.  In contrast, there was no significant change in the morphology in the non-exposed iRBC at all concentrations and exposure time.  This observation was in agreement with the levels of the enzymatic degradation products iron and bilirubin, of which the highest levels (106.03 and 1,753.54% of baseline level, respectively) were observed at 15 vs. 20 M at 3 vs. 24 hours exposure.  For the effect of the HO-1 inhibitor ZnPPIX, HBMEC cell morphology was mostly unchanged, but significant inhibitory effect on cell apoptosis was seen at 10 M for the exposure period of 3 hours (37.17% of baseline level).  The degree of the inhibitory effect as reflected by the level of iron produced was not clearly observed (highest effect at 10 M and 3 hours exposure). Results provide at least in part, insight into the contribution of HO-1 on CM pathogenesis and need to be confirmed in animal model. The role of prostaglandin D2 (PGD2) in malaria pathogenesis and severity was investigated in 120 blood and urinary samples collected from Thai patients with      P. falciparum with uncomplicated (n=26) and complicated (n=4), patients with         P. vivax (n=30), patients with fever associated with other infections (n=30), and healthy subjects (n=30).  PGD2 concentrations in plasma and urinary samples were determined using Prostaglandin D2-MOX express EIA kit (Cayman Chemical, USA).  The possible association between PGD2 and malaria infections was clearly demonstrated with urinary PGD2 concentrations.  The urinary PGD2 concentrations were relatively high (about 5-fold) in patients with P. falciparum with uncomplicated and P. vivax infections compared with other groups.  Furthermore, the concentration in patients with P. falciparum with uncomplicated and P. vivax infection were significantly higher than that in healthy subjects and patients with fever associated with other infections.  Urinary PGD2 concentrations may offer a more dependable and useful tool for predicting malaria severity.  The diagnostic performance of nested PCR and a recently developed an ELISA-based new rapid diagnosis test (RDT), NovaLisa test kit, for diagnosis of malaria infection, was investigated using microscopic method as the gold standard.  The performance of nested-PCR as a malaria diagnostic tool is excellent with respect to its high accuracy, sensitivity, specificity, and ability to discriminate Plasmodium species.  The sensitivity and specificity of nested-PCR compared with the microscopic method for detection of P. falciparum, P. vivax, and P. falciparum/P. vivax mixed infection were 71.4 vs 100%, 100 vs 98.7%, and 100 vs 95.0%, respectively.  The sensitivity and specificity of the ELISA-based NovaLisa test kit compared with the microscopic method for detection of Plasmodium genus were 89.0 vs 91.6%, respectively.  NovaLisa test kit provided comparable diagnostic performance.  Its relatively low cost, simplicity, and rapidity enables large scale field application. The analysis of prevalence and distribution of  P. vivax dihydrofolate reductase (pvdhfr) and P. vivax dihydropteroate synthase (pvdhps) mutations were performed in 169 samples collected from patients with P. vivax infection who attended the malaria clinics in the provinces along the three international borders of Thailand (Thai-Myanmar, Thai-Cambodian, and Thai-Malaysian borders).  SNP-haplotypes of the pvdhfr at amino acid positions 13, 33, 57, 58, 61, 117, and 173 and of the pvdhps at positions 383 and 553 were examined by nested PCR-RFLP.  Signiﬁcant differences in the prevalence and distribution of pvdhfr and pvdhps combination alleles were observed in P. vivax isolates collected from all the three border areas.  The most prevalent combination alleles were triple mutant pvdhfr 57L/58R/117T alleles/double wild-type pvdhps alleles (n = 18), double mutant pvdhfr 58R/117N alleles/double wild-type pvdhps alleles (n = 10), and triple mutant pvdhfr 58R/61M/117N alleles/double wild-type pvdhps alleles (n = 52) or with single mutant pvdhps 383G allele (n = 28), respectively.  This information on prevalence and patterns of pvdhfr and pvdhps polymorphisms obtained from the present study suggests the presence of SP pressure on P. vivax isolates in Thailand which could be linked to the introduction of malaria from neighboring countries. Results did not support the application of SP for P. vivax control program in Thailand as well as the neighboring countries. The genetic polymorphisms of pvdhfr and pvdhps genes of P. vivax were also investigated in 83 blood samples collected from patients in the Philippines, Bangladesh, and Nepal.  The SNP-haplotypes of the pvdhfr gene at the amino acid positions 13, 33, 57, 58, 61, 117, and 173, and that of the pvdhps gene at the positions 383 and 553 were analyzed by nested PCR-RFLP.  Results suggest diverse polymorphic patterns of pvdhfr alone as well as the combination patterns with pvdhps mutant alleles in P. vivax isolates collected from the 3 endemic countries in Asia.  All samples carried mutant combination alleles of pvdhfr and pvdhps.  The most prevalent combination alleles found in samples from the Philippines and Bangladesh were triple mutant pvdhfr combined with single mutant pvdhps allele and triple mutant pvdhfr combined with double wild-type pvdhps alleles, respectively.  Those collected from Nepal were quadruple mutant pvdhfr combined with double wild-type pvdhps alleles.  New alternative antifolate drugs which are effective against sulfadoxine-pyrimethamine (SP)-resistant P. vivax are required.The sensitivity of ten P. vivax isolates to chloroquine and P. falciparum isolates to chloroquine, quinine and artemisinin collected from Mae Sot district, Tak province in 2012, was investigated in association with polymorphisms of the parasite resistant genes, i.e., P. vivax chloroquine resistance transporter (pvcrt-o), P. vivax multidrug resistance transporter (pvmdr1), P. falciparum multidrug resistance protein 1 (pfmrp1: SNP at amino acid positions 191 and 437), P. falciparum sodium hydrogen exchanger 1 (pfnhe1: at the microsatellite in ms4760 locus), P. falciparum maurer’s cleft two transmembrane proteins (pfmc-2tm: new SNPs), and P. falciparum GTP cyclohydrolase 1 (pfgch1: new SNPs).  Results revealed non-synonymous mutations at position 196 of pvcrt-o.  For pvmdr1, non-synonymous mutations at position 976 and 1076.   Non-synonymous mutations at the position 437 of pfmrp1 were found.  For pfnhe1, the amino acids were NHNDNHNNDDD repeat and DNNND repeat.  For pfmc-2tm, non-synonymous mutations at the positions 189, 190, 192, 194, 196 and 197 were found.  All isolates carried wild type pfgch1.  These genetic markers should be further validated for monitoring of antimalarial drug resistance.