sybr-green-i and Malaria

As an alternative to PCR methods, LAMP is increasingly being used in the field of molecular diagnostics. Under isothermal conditions at 65 °C, the entire procedure takes approximately 30 min to complete. In this study, we establish a sensitive and visualized LAMP method in a closed-tube system for the detection of Plasmodium knowlesi.. A total of 71 malaria microscopy positive blood samples collected in blood spots were obtained from the Sarawak State Health Department. Using 18s rRNA as the target gene, nested PCR and SYBR green I LAMP assay were performed following the DNA extraction. The colour changes of LAMP end products were observed by naked eyes.. LAMP assay demonstrated a detection limit of 10 copies/µL in comparison with 100 copies/µL nested PCR. Of 71 P. knowlesi blood samples collected, LAMP detected 69 microscopy-positive samples. LAMP exhibited higher sensitivity than nested PCR assay. The SYBR green I LAMP assay was 97.1% sensitive (95% CI 90.2-99.7%) and 100% specific (95% CI 83.2-100%). Without opening the cap, incorporation of SYBR green I into the inner cap of the tube enabled the direct visualization of results upon completion of amplification. The positives instantaneously turned green while the negatives remained orange.. These results indicate that SYBR green I LAMP assay is a convenient diagnosis tool for the detection of P. knowlesi in remote settings.

Topics: Benzothiazoles; Diamines; Humans; Malaria; Malaysia; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Plasmodium knowlesi; Quinolines; RNA, Protozoan; RNA, Ribosomal, 18S; Sensitivity and Specificity

The Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding. Several molecular diagnostic techniques for distinguishing between An. coluzzii and An. gambiae are still under improvement. Although, the current SINE method for identification between An. coluzzii and An. gambiae works reliably, this study describes a refinement of the SINE method to increase sensitivity for identification of An. coluzzii, An. gambiae and An. arabiensis based on amplicon dissociation curve characteristics. Field-collected samples, laboratory-reared colonies and crossed specimens of the two species were used for the design of the protocol. An. gambiae, An. coluzzii, and hybrids of the two species were sampled from Ghana and An. arabiensis from Kenya. Samples were first characterised using conventional SINE PCR method, and further assayed using SYBR green, an intercalating fluorescent dye. The three species and hybrids were clearly differentiated using the melting temperature of the dissociation curves, with derivative peaks at 72°C for An. arabiensis, 75°C for An. gambiae and 86°C for An. coluzzii. The hybrids (An. gambiae / An. coluzzii) showed both peaks. This work is the first to describe a SYBR green real time PCR method for the characterization of An. arabiensis, An. gambiae and An. coluzzii and was purposely designed for basic melt-curve analysis (rather than high-resolution melt-curve) to allow it to be used on a wide range of real-time PCR machines.

Topics: Animals; Anopheles; Benzothiazoles; Diamines; DNA; Fluorescent Dyes; Ghana; Kenya; Malaria; Molecular Diagnostic Techniques; Mosquito Vectors; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Reproducibility of Results; Sensitivity and Specificity; Species Specificity

In epidemiological surveys and surveillance the application of molecular tools is essential in detecting submicroscopic malaria. A genus-specific conventional cytochrome b (cytb) PCR has shown high sensitivity in field studies, detecting 70% submicroscopic malaria. The main objective of this study was to assess the conversion from conventional to real-time PCR testing both SYBR and probe protocols, and including quantitative (q) PCR. The protocols were assessed applying well-defined clinical patient material consisting of 33 positive and 80 negative samples. Sequencing of positive PCR products was performed. In addition, a sensitivity comparison of real-time PCR methods was done by including five relevant assays investigating the effect of amplification target and platform. Sensitivity was further examined using field material consisting of 111 P.falciparum positive samples from Tanzanian children (< 5 years), as well as using related patient data to assess the application of q-PCR with focus on low-level parasitaemia. Both the cytb SYBR and probe PCR protocols showed as high sensitivity and specificity as their conventional counterpart, except missing one P. malariae sample. The SYBR protocol was more sensitive and specific than using probe. Overall, choice of amplification target applied is relevant for achieving ultra-sensitivity, and using intercalating fluorescence dye rather than labelled hydrolysis probes is favourable. Application of q-PCR analysis in field projects is important for the awareness and understanding of low-level parasitaemia. For use in clinical diagnosis and epidemiological studies the highly sensitive and user-friendly cytb SYBR q-PCR method is a relevant tool. The genus-specific method has the advantage that species identification by sequencing can be performed as an alternative to species-specific PCR.

Topics: Benzothiazoles; Child, Preschool; Cytochromes b; Diamines; DNA, Protozoan; Genes, Protozoan; Humans; Infant; Infant, Newborn; Malaria; Organic Chemicals; Parasitemia; Plasmodium falciparum; Quinolines; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Sensitivity and Specificity

The in vivo efficacy of potential antimalarials is usually evaluated by direct microscopic determination of the parasitaemia of Plasmodium-infected mice on Giemsa-stained blood smears. This process is time-consuming, requires experienced technicians and is not automatable. Therefore, we optimized a SYBR Green I (SYBRG I) fluorescence-based assay to fluorometers commonly available in many research laboratories. This technique was originally developed to assess parasitaemia in humans by cytometry. We defined optimal conditions with Plasmodium berghei-infected mice, standard lysis buffer (Tris, EDTA, saponin and Triton), whole blood cells and 2 h staining incubation with SYBRG I 2X. The fluorescence background generated by uninfected whole blood cells was low (around 4.6%), and the linearity high (r

Topics: Animals; Antimalarials; Azure Stains; Benzothiazoles; Diamines; Fluorescence; Malaria; Male; Mice; Microbial Sensitivity Tests; Organic Chemicals; Plasmodium berghei; Quinolines

The simian malaria parasite Plasmodium knowlesi is now a well-recognized pathogen of humans in South-East Asia. Clinical infections appear adequately treated with existing drug regimens, but the evidence base for this practice remains weak. The availability of P. knowlesi cultures adapted to continuous propagation in human erythrocytes enables specific studies of in vitro susceptibility of the species to antimalarial agents, and could provide a surrogate system for testing investigational compounds against Plasmodium vivax and other non-Plasmodium falciparum infections that cannot currently be propagated in vitro.. We sought to optimize protocols for in vitro susceptibility testing of P. knowlesi and to contrast outputs with those obtained for P. falciparum under comparable test conditions.. Growth monitoring of P. knowlesi in vitro was by DNA quantification using a SYBR Green fluorescent assay or by colorimetric detection of the lactate dehydrogenase enzyme. For comparison, P. falciparum was tested under conditions identical to those used for P. knowlesi.. The SYBR Green I assay proved the most robust format over one (27 h) or two (54 h) P. knowlesi life cycles. Unexpectedly, P. knowlesi displays significantly greater susceptibility to the dihydrofolate reductase inhibitors pyrimethamine, cycloguanil and trimethoprim than does P. falciparum, but is less susceptible to the selective agents blasticidin and DSM1 used in parasite transfections. Inhibitors of dihydroorotate dehydrogenase also demonstrate lower activity against P. knowlesi.. The fluorescent assay system validated here identified species-specific P. knowlesi drug susceptibility profiles and can be used for testing investigational compounds for activity against non-P. falciparum malaria.

Topics: Antimalarials; Benzothiazoles; Colorimetry; Diamines; Dihydroorotate Dehydrogenase; Erythrocytes; Fluorescence; Humans; L-Lactate Dehydrogenase; Malaria; Organic Chemicals; Oxidoreductases Acting on CH-CH Group Donors; Parasitic Sensitivity Tests; Plasmodium falciparum; Plasmodium knowlesi; Proguanil; Pyrimethamine; Quinolines; Sensitivity and Specificity; Triazines

The erythrocytic life cycle of Plasmodium falciparum is highly associated with severe clinical symptoms of malaria that causes hundreds of thousands of death each year. The parasite develops within human erythrocytes leading to the disruption of the infected red blood cell (iRBC) prior to the start of a new cycle of erythrocyte infection. Emerging mechanisms of resistance against antimalarial drugs require improved knowledge about parasite's blood stages to facilitate new alternative antimalarial strategies. For the analysis of young blood stages of Plasmodium at the molecular level, the isolation of ring stages is essential. However, early stages can hardly be separated from both, late stages and non-infected red blood cells using conventional methods. Here, iRBCs were stained with the DNA-binding dyes Vybrant® DyeCycle™ Violet and SYBR® Green I. Subsequently, cells were subjected to flow-cytometric analysis. This enabled the discrimination of early stage iRBCs as well as late-stage iRBCs from non-infected erythrocytes and the properties of the used dyes were evaluated. Moreover, early stage iRBCs were isolated with high purity (>98%) by FACS. Subsequently, development of sorted early stages of the parasite was monitored over time and compared with control cultures. The described flow cytometry method, based on staining with Vybrant DyeCycle Violet, allows the isolation of viable ring stages of the malarial agent P. falciparum, and thereby provides the basis for new, broad-range molecular investigations of the parasite.

Topics: Azure Stains; Benzothiazoles; Diamines; Erythrocytes; Flow Cytometry; Fluorescent Dyes; Humans; Malaria; Organic Chemicals; Parasitemia; Plasmodium falciparum; Quinolines; Schizonts; Staining and Labeling; Time Factors

Recently, Plasmodium knowlesi has been recognised as the fifth Plasmodium species causing malaria in humans. Hundreds of human cases infected with this originally simian Plasmodium species have been described in Asian countries and increasing numbers are reported in Europe from travellers. The growing impact of tourism and economic development in South and Southeast Asia are expected to subsequently lead to a further increase in cases both among locals and among travellers. P. knowlesi is easily misidentified in microscopy as P. malariae or P. falciparum. We developed new primers for the rapid and specific detection of this species by low-cost real-time polymerase chain reaction (PCR) and added this method to an already existing panel of primers used for the molecular identification of the other four species in one reaction. Reference laboratories should now be able to identify undisputably and rapidly P. knowlesi, as it is a potentially fatal pathogen.

Topics: Animals; Benzothiazoles; Diamines; DNA Primers; DNA, Protozoan; Europe; Humans; Malaria; Organic Chemicals; Plasmodium; Plasmodium knowlesi; Polymerase Chain Reaction; Quinolines; Sensitivity and Specificity; Species Specificity; Travel