ABSTRACT
Genetic polymorphisms in the malaria parasite Plasmodium falciparum mediate alterations in sensitivity to important antimalarial drugs. Surveillance for these polymorphisms is helpful in assessing the prevalence of drug resistance and designing strategies for malaria control. Multiple methods are available for the assessment of P. falciparum genetic polymorphisms, but they suffer from low throughput, technical limitations, and high cost. We have optimized and tested a multiplex ligase detection reaction-fluorescent microsphere (LDR-FM) assay for the identification of important P. falciparum genetic polymorphisms. For 84 clinical samples from Kampala, Uganda, a region where both transmission intensity and infection complexity are high, DNA was extracted from dried blood spots, genes of interest were amplified, amplicons were subjected to multiplex ligase detection reactions to add bead-specific oligonucleotides and biotin, fragments were hybridized to magnetic beads, and polymorphism prevalences were assessed fluorometrically in a multiplex format. A total of 19 alleles from the pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps genes were analyzed by LDR-FM and restriction fragment length polymorphism (RFLP) analyses. Considering samples with results from the two assays, concordance between the assays was good, with 78 to 100% of results identical at individual alleles, most nonconcordant results differing only between a mixed and pure genotype call, and full disagreement at individual alleles in only 0 to 3% of results. We estimate that the LDR-FM assay offers much higher throughput and lower cost than RFLP. Our results suggest that the LDR-FM system offers an accurate high-throughput means of classifying genetic polymorphisms in field samples of P. falciparum.
Subject(s)
Drug Resistance , Molecular Diagnostic Techniques/methods , Plasmodium falciparum/genetics , Polymorphism, Genetic , Antimalarials/pharmacology , Blood/parasitology , High-Throughput Screening Assays/economics , High-Throughput Screening Assays/methods , Humans , Ligases/metabolism , Malaria, Falciparum/parasitology , Microspheres , Molecular Diagnostic Techniques/economics , Parasitic Sensitivity Tests/economics , Parasitic Sensitivity Tests/methods , Plasmodium falciparum/drug effects , Plasmodium falciparum/isolation & purification , UgandaABSTRACT
Simple, cost-effective approach for routine surveillance of parasite susceptibility to antileishmanial drug miltefosine (MIL) is highly desirable for controlling emergence of drug resistance in visceral leishmaniasis (VL). We validated a simple resazurin-based fluorimetric assay using promastigotes to track natural MIL tolerance in Leishmania donovani parasites from VL cases (n = 17) against standard amastigote assay, in two different labs in India. The inter-stage MIL susceptibility correlated strongly (r = 0.70, p = 0.0018) using J774.A.1 macrophage cell line-based amastigote assay and fluorescence-based resazurin assay for promastigotes. Investigation of inter-stage MIL susceptibility for the same set of clinical isolates in another lab also showed a strong correlation (r = 0.72, p = 0.0012) using mouse peritoneal macrophages for amastigote assay and resazurin-based alamar blue assay for promastigotes. Additionally, parasites from post-kala-azar dermal leishmaniasis (PKDL) lesions (n = 7, r = 0.78, p = 0.046) and MIL-induced parasites (r = 0.92, p = 0.0001; n = 3) also exhibited a strongly correlated inter-stage miltefosine susceptibility. Thus, our results support the utility of resazurin assay as a simplified biological tool for MIL susceptibility monitoring in clinical isolates from MIL-treated VL/PKDL patients.
Subject(s)
Antiprotozoal Agents/pharmacology , Fluorometry/methods , Leishmania donovani/drug effects , Phosphorylcholine/analogs & derivatives , Animals , Cell Line , India , Macrophages/parasitology , Mice , Oxazines/metabolism , Parasitic Sensitivity Tests/economics , Parasitic Sensitivity Tests/methods , Phosphorylcholine/pharmacology , Staining and Labeling/methods , Xanthenes/metabolismABSTRACT
Drug infused mini agar plates were found to be a better alternative of broth dilution method in the determination of antileishmanial susceptibility of two commonly used drugs, Sodium antimony gluconate and Amphotericin B against Leishmania donovani promastigotes. These two drugs were used here as models for antileishmanial compounds. The stability of the drugs in the stored agar plates was also tested for six months and found that they were same as fresh plates. Determination of antileishmanial susceptibility of Leishmania donovani promastigotes to compounds of screening by this method is quite inexpensive, simple to perform even in under-sophisticated laboratories of developing countries where the disease is endemic.
Subject(s)
Antiprotozoal Agents/pharmacology , Leishmania donovani/drug effects , Agar , Amphotericin B/pharmacology , Antimony Sodium Gluconate/pharmacology , Culture Media/chemistry , Drug Stability , Humans , Parasitic Sensitivity Tests/economics , Parasitic Sensitivity Tests/methodsABSTRACT
Isolation and propagation of Leishmania parasites using conventional culture methods are difficult, especially under field conditions. Transportation of live parasites requires the maintenance of low temperatures, which increases the cost considerably. The present study demonstrates that monophasic micro capillary culture is a simpler, more economical and better alternative to using conventional Evan's modified Tobie's medium to isolate and transport the Leishmania parasite over long distances without the use of temperature control methods.
