Assessment of the Worldwide Antimalarial Resistance Network Standardized Procedure for In Vitro Malaria Drug Sensitivity Testing Using SYBR Green Assay for Field Samples with Various Initial Parasitemia Levels

Abstract

The malaria SYBR green assay, which is used to profile in vitro drug susceptibility of Plasmodium falciparum, is a reliable drug screening and surveillance tool. Malaria field surveillance efforts provide isolates with various low levels of parasitemia. To be advantageous, malaria drug sensitivity assays should perform reproducibly among various starting parasitemia levels rather than at one fixed initial value. We examined the SYBR green assay standardized procedure developed by the Worldwide Antimalarial Resistance Network (WWARN) for its sensitivity and ability to accurately determine the drug concentration that inhibits parasite growth by 50% (IC 50 ) in samples with a range of initial parasitemia levels. The initial sensitivity determination of the WWARN procedure yielded a detection limit of 0.019% parasitemia. P. falciparum laboratory strains and field isolates with various levels of initial parasitemia were then subjected to a range of doses of common antimalarials. The IC 50 s were comparable for laboratory strains with between 0.0375% and 0.6% parasitemia and for field isolates with between 0.075% and 0.6% parasitemia for all drugs tested. Furthermore, assay quality (Z=) analysis indicated that the WWARN procedure displays high robustness, allowing for drug testing of malaria field samples within the derived range of initial parasitemia. The use of the WWARN procedure should allow for the inclusion of more malaria field samples in malaria drug sensitivity screens that would have otherwise been excluded due to low initial parasitemia levels.

Real-time cholesterol sorting in Plasmodium falciparum-erythrocytes as revealed by 3D label-free imaging

“…SYBR green assays. SYBR green assays were performed by modifying the methods from previous studies 46,63,64 for evaluating P. falciparum growth. Briefly, P. falciparum was cultured for ≈96 h (two cycles), the P. falciparum-RBC burst was collected in sterilized water, and the erythrocyte membranes were removed by centrifugation (18,000 × g, 15 min).…”

The Role of Complement Immune Response on Artemisinin Combination Therapy in a Population from Malaria Endemic Region of Western Kenya

“…Parasite growth was evaluated using SYBR Green I dye, thus 4 µL of SYBR Green I was added to 2 mL of malaria SYBR Green 1 fluorescence (MSF) lysis buffer [20 mM Tris (pH 7.5), 5 mM EDTA, 0.16% (wt/vol) saponin, and 1.6% (vol/vol) Triton X-100], 10× _ SYBR green I dye concentration) and 100 µL of a mixture of SYBR Green 1 and MSF was transferred to each well. The plates were stored in the dark at ambient temperature for 24 hours [18]. The plates were examined for relative fluorescence units (RFUs) per well using a fluorescence plate reader (Tecan™ Morrisville, North Carolina, United States).…”

Methods for monitoring artemisinin-based combination therapies efficacy

“…The geometric mean IC 50 values from SYBR Green 1-based ex-vivo study of susceptibility of Ghanaian P. falciparum clinical isolates to a panel of anti-malarial drugs were below the resistance threshold for atovaquone (ATV), AS, DHA, artemether (ATM), LM, AQ, MQ, PQP and CQ, respectively . SYBR Green 1-based ex-vivo study of susceptibility of laboratory culture adapted parasite line D6 and clinical isolates from the Worldwide Antimalarial Resistance Network with various initial parasitemia levels were all sensitive to ATV, ART, MQ and CQ (Cheruiyot et al, 2016).…”

“…Because of biosafety concerns with radioactive reagents, and the high cost of equipment and reagents, fluorometric and ELISA methods were developed to monitor ACTs partner drugs. The main fluorophore SYBR Green I-based methods were used in several studies (Cheruiyot et al, 2016;Lim et al, 2013;Quashie et al, 2013). Two enzyme-linked immunosorbent assay (ELISA)-based methods that use mono-clonal antibodies to Plasmodium spp.…”

“…In vitro/ex-vivo fluorometric and ELISA based tests Kaddouri et al (2008), Fall et al (2011), Quashie et al (2013), Cheruiyot et al (2016), Noedl et al (2005) and Bacon et al (2007) IC 50 obtained from staining with Malaria SYBR Green 1 fluorescent give high noise background. The limitation of this method is the non-specificity of this dye that can also stain human white blood cells.…”