Molecular diagnosis of drug resistance in Plasmodium falciparum and virulence factors in Entamoeba histolytica

Abstract

Four species of the protozoan parasite genus Plasmodium are known to infect man. Plasmodium falciparum, the most virulent species is increasingly associated with resistance to antimalarial drugs. Drug resistance to one group of antimalarials, the antifolates pyrimethamine and cycloguanil has been associate with specific mutations in the dihydrofolate reductase (dhfr) gene of the folic acid cycle of the malaria parasite corresponding to amino acid changes in the DHFR protein.

The DNA sequences of the dhfr gene of P. falciparum were studied in Vietnamese isolates. Most of isolates showed a cycloguanil sensitive profile while having a resistant pyrimethamine profile. Only one isolate showed a resistant profile to both antifolates. A novel mutation Leu-140 was found in one Vietnamese isolate and in one Thai clone. These parasites also had the Val-16 and Thr-108 mutations, associated with cycloguanil resistance.

A new PCR-restriction-enzyme method was developed to detect pyrimethamine and/or cycloguanil resistance of P. falciparum, based on restriction enzyme Alu 1 cleavage of the PCR product from the dhfr gene. The enzyme cleaves the dhfr gene when there is a serine amino acid at position 108 whereas any single point mutation leaves the DNA uncleaved.

Several species of the protozoan parasite genus Entamoeba infect humans, but only Entamoeba histolytica causes disease. E. histolytica parasites colonize the human host and produce factors involved in tissue destruction. Two previously described hemolysins, HLY 1 and HLY4 were mapped to the ribosomal DNA of E. histolytica. In addition, one novel hemolysin gene HLY6 was identified located on the antisense strand of the large subunit (Isu) of the rRNA gene.

A new PCR assay (hemo-PCR) based on the novel E. histolytica hemolysin gene HLY6 was also developed and evaluated on different Entamoeba species, amoebic liver abscesses (ALA), stool sample. human and bacterial DNA by cleavage of the PCR products with Alu 1.

Defined open reading frames (ORF) were designed for each of the three different gene products by the use of gene-fusion technology and frame shift mutation. The hemolytic activity was only expressed when the appropriate ORF's were in the correct reading frame. Introduction of non-sense mutations in the HLY4 gene completely abolished hemolytic activity whereas introduction of the mutated alleles into Escherichia coli suppressor strains restored the activity.

All three hemolysins reacted with antisera from patients with invasive amoebiasis in a modified ELISA test. A band representing the hemolysin HLY4 in Western-blot analysis was detected. An assay was therefore developed for the specific detection of E. histolytica infection, based on HLY4-hemolysis-inhibition, in a dose dependent fashion using sera from amoebiasis patients. The hemo-PCR and the HLY4-hemolysis-inhibition assays represent specific diagnostic tools for the identification of E. histolytica infection.