ABSTRACT
Malaria morbidity and mortality have decreased gradually in the Greater Mekong Subregion (GMS). Presently, WHO sets a goal to eliminate malaria by 2030 in the GMS. However, drug-resistant malaria has been reported from several endemic areas. To achieve the goal of elimination, the status of the emergence and spread of drug resistance should be monitored. In this study, the genotype of the Plasmodium falciparum chloroquine (CQ) resistance transporter gene (pfcrt) and 6 microsatellite DNA loci flanking the gene were examined. P. falciparum isolates (nâ¯=â¯136) was collected from malaria patients in Thailand (nâ¯=â¯50, 2002-2005), Vietnam (nâ¯=â¯39, 2004), Laos (nâ¯=â¯15, 2007) and Cambodia (nâ¯=â¯32, 2009). Amino acid sequences at codons 72-76 on the gene were determined. All of the isolates from Thailand were CQ-resistant (CVIET), as were all of the isolates from Cambodia (CVIET, CVIDT). Thirteen of the 15 isolates (87%) from Laos were CQ-resistant (CVIET, CVIDT), whereas the other 2 (13%) were CQ-susceptible (CVMNK). In contrast, 27 of the 39 isolates (69%) from Vietnam were CQ-susceptible (CVMNK), whereas the other 12 (31%) were CQ-resistant (CVIET, CVIDT, CVMDT) or mixed (CVMNK/CVIDT). The mean of expected heterozygosity of the microsatellite loci was 0.444 in the Thai population, 0.482 in the Cambodian population, and 0.734 in the Vietnamese population. Genetic diversity in the Thai population was significantly lower than that in the Vietnamese population. These results suggested that chloroquine selective pressure on P. falciparum populations is heterogeneous in the GMS. Therefore, further examination to understand the mechanisms behind the emergence and spread of drug-resistant malaria are needed.
Subject(s)
DNA, Protozoan/genetics , Genotype , Membrane Transport Proteins/genetics , Microsatellite Repeats/genetics , Plasmodium falciparum/genetics , Polymorphism, Genetic , Protozoan Proteins/genetics , Antimalarials/pharmacology , Asia, Southeastern , Chloroquine/pharmacology , Drug Resistance , MutationABSTRACT
The detection of gametocytes in human peripheral blood is one of the most important measures in a malaria survey. We attempted to detect gametocytes of Plasmodium falciparum by reverse transcription polymerase chain reaction (RT-PCR) of dried blood on filter paper. On field samples analysis, the specific RT-PCR products for region 3 of pfg377 mRNA were observed in 67 of 131 falciparum malaria patients. The minimum detection level of RT-PCR-positive samples was 0.03 gametocytes/microl on quantitative real-time RT-PCR. Gametocyte positive rate was not dependent on sex or age. A higher frequency of gametocytes was found in single P. falciparum infection than in mixed species infection (P<0.01). In this study, 47 of the 131 patients were asymptomatic. Eighteen of these 47 patients showed pfg377 mRNA expression. Moreover, four alleles of region 3 of pfg377 were detected in pfg377 mRNA-positive patients and 13 of 67 pfg377 mRNA-positive patients carried more than one gametocyte-producing clone. These results suggest that dried blood on filter paper is a useful for a molecular epidemiologic study of malaria transmission and gametocyte-targeted control.
Subject(s)
Blood Specimen Collection/methods , Plasmodium falciparum/growth & development , Reverse Transcriptase Polymerase Chain Reaction/methods , Adolescent , Adult , Aged , Amino Acid Sequence , Animals , Blood Specimen Collection/instrumentation , Child , Child, Preschool , Female , Filtration/instrumentation , Humans , Infant , Malaria, Falciparum/diagnosis , Malaria, Falciparum/epidemiology , Malaria, Falciparum/parasitology , Male , Middle Aged , Molecular Epidemiology , Molecular Sequence Data , Paper , Plasmodium falciparum/genetics , Plasmodium falciparum/isolation & purification , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, DNAABSTRACT
We examined the role of osteopontin (OPN) in immunity against Plasmodium falciparum infection. We measured the mRNA levels for OPN and several cytokines in RNA preparations extracted from dried blood on filter paper obtained from falciparum malaria patients in Vietnam. Expression of OPN mRNA was detected in 134 of 161 patients. The expression of both interleukin-12 p40 and interferon-gamma mRNAs in the group positive for OPN mRNA was significantly higher than that in the group negative for OPN mRNA. The level of parasitemia in the OPN mRNA-positive group was much lower than that in the negative one. These results suggest that OPN might suppress multiplication of the parasites through T helper 1 cells-mediated immune responses.
