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A multiplex ligase detection reaction-fluorescent microsphere assay for simultaneous detection of single nucleotide polymorphisms associated with Plasmodium falciparum drug resistance.

ABSTRACT: Incomplete malaria control efforts have resulted in a worldwide increase in resistance to drugs used to treat the disease. A complex array of mutations underlying antimalarial drug resistance complicates efficient monitoring of parasite populations and limits the success of malaria control efforts in regions of endemicity. To improve the surveillance of Plasmodium falciparum drug resistance, we developed a multiplex ligase detection reaction-fluorescent-microsphere-based assay (LDR-FMA) that identifies single nucleotide polymorphisms (SNPs) in the P. falciparum dhfr (9 alleles), dhps (10 alleles), and pfcrt (3 alleles) genes associated with resistance to Fansidar and chloroquine. We evaluated 1,121 blood samples from study participants in the Wosera region of Papua New Guinea, where malaria is endemic. Results showed that 468 samples were P. falciparum negative and 453 samples were P. falciparum positive by a Plasmodium species assay and all three gene assays (concordance, 82.2%). For P. falciparum infections where the assay for each gene was positive, 2 samples carried resistance alleles for all three genes, 299 carried resistance alleles for dhfr and pfcrt, 131 carried resistance alleles for only one gene (dhfr [n = 40], dhps [n = 1], or pfcrt [n = 90]), and 21 carried only sensitive alleles at all three genes. Mixed-strain infections characterized 100 samples. Overall, 95.4% (432/453) of P. falciparum-infected samples carried at least one allele associated with resistance to Fansidar or chloroquine. In view of the fact that 86.3% (391/453) of P. falciparum-infected samples carried pfcrt mutations, chloroquine is largely ineffective against P. falciparum in Papua New Guinea. Surveillance of additional dhfr and dhps polymorphisms in order to monitor the continued effectiveness of Fansidar is recommended.

SUBMITTER: Carnevale EP

PROVIDER: S-EPMC1829096 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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A multiplex ligase detection reaction-fluorescent microsphere assay for simultaneous detection of single nucleotide polymorphisms associated with Plasmodium falciparum drug resistance.

Carnevale Eric P EP Kouri Drew D DaRe Jeana T JT McNamara David T DT Mueller Ivo I Zimmerman Peter A PA

Journal of clinical microbiology 20061122 3

Incomplete malaria control efforts have resulted in a worldwide increase in resistance to drugs used to treat the disease. A complex array of mutations underlying antimalarial drug resistance complicates efficient monitoring of parasite populations and limits the success of malaria control efforts in regions of endemicity. To improve the surveillance of Plasmodium falciparum drug resistance, we developed a multiplex ligase detection reaction-fluorescent-microsphere-based assay (LDR-FMA) that ide ...[more]

PMID: 17121999

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Project description:BackgroundAssessment exposure and immunity to malaria is an important step in the fight against the disease. Increased malaria infection in non-immune travellers under anti-malarial chemoprophylaxis, as well as the implementation of malaria elimination programmes in endemic countries, raises new issues that pertain to these processes. Notably, monitoring malaria immunity has become more difficult in individuals showing low antibody (Ab) responses or taking medications against the Plasmodium falciparum blood stages. Commonly available techniques in malaria seroepidemiology have limited sensitivity, both against pre-erythrocytic, as against blood stages of the parasite. Thus, the aim of this study was to develop a sensitive tool to assess the exposure to malaria or to bites from the vector Anopheles gambiae, despite anti-malarial prophylactic treatment.MethodsAb responses to 13 pre-erythrocytic P. falciparum-specific peptides derived from the proteins Lsa1, Lsa3, Glurp, Salsa, Trap, Starp, CSP and Pf11.1, and to 2 peptides specific for the Anopheles gambiae saliva protein gSG6 were tested. In this study, 253 individuals from three Senegalese areas with different transmission intensities and 124 European travellers exposed to malaria during a short period of time were included.ResultsThe multiplex assay was optimized for most but not all of the antigens. It was rapid, reproducible and required a small volume of serum. Proportions of Ab-positive individuals, Ab levels and the mean number of antigens (Ags) recognized by each individual increased significantly with increases in the level of malaria exposure.ConclusionThe multiplex assay developed here provides a useful tool to evaluate immune responses to multiple Ags in large populations, even when only small amounts of serum are available, or Ab titres are low, as in case of travellers. Finally, the relationship of Ab responses with malaria endemicity levels provides a way to monitor exposure in differentially exposed autochthonous individuals from various endemicity areas, as well as in travellers who are not immune, thus indirectly assessing the parasite transmission and malaria risk in the new eradication era.

Project description:BackgroundMultiplex cytokine profiling systems are useful tools for investigating correlates of protective immunity. Several Luminex and flow cytometry methods are commercially available but there is limited information on the relative performance of different kits. A series of comparison experiments were carried out to determine the most appropriate method for our subsequent studies.MethodsTwo Luminex methods were compared, the Bio-Rad human 17-plex panel and the Invitrogen (formerly BioSource) human cytokine 10-plex kit, and two flow cytometry methods, the Becton Dickinson Human Th1/Th2 Cytokine Kit (CBA) and the Bender MedSystems Human Th1/Th2 11plex FlowCytomix Multiplex Kit. All kits were tested for the measurement of cytokines in supernatants collected from human leukocytes stimulated with viable Plasmodium falciparum infected red blood cells (iRBC) or P. falciparum schizont lysates.ResultsData indicated that the kits differed in sensitivity and reproducibility depending on the cytokine, and detected different quantities of some cytokines. The Bio-Rad 17-plex kit was able to detect more positive responses than the Invitrogen 10-plex kit. However, only when detecting IL-1, IL-6 or TNF did the two Luminex based methods correlate with one another. In this study, the flow cytometry based techniques were less variable and correlated better with one another. The two flow cytometry based kits showed significant correlation when detecting IFN-?, IL-2, TNF, IL-10 and IL-6, but overall the BD kit detected more positive responses than the Bender MedSystems kit.ConclusionsThe microsphere suspension array technologies tested differed in reproducibility and the absolute quantity of cytokine detected. Sample volume, the number of cytokines measured, and the time and cost of the assays also differed. These data provide an accurate assessment of the four techniques, which will allow individual researchers to select the tool most suited for their study population.

Dataset Information

A multiplex ligase detection reaction-fluorescent microsphere assay for simultaneous detection of single nucleotide polymorphisms associated with Plasmodium falciparum drug resistance.

Publications

A multiplex ligase detection reaction-fluorescent microsphere assay for simultaneous detection of single nucleotide polymorphisms associated with Plasmodium falciparum drug resistance.

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