Antimalarial Drug Sensitivity Profile of Western Kenya Plasmodium falciparum Field Isolates Determined by a SYBR Green I in vitro Assay and Molecular Analysis

Akala, Hoseah M.; Eyase, Fredrick; Cheruiyot, Agnes C.; Omondi, Angela A.; Ogutu, Bernhards; Waters, Norman C.; Johnson, Jacob; Polhemus, Mark E.; Schnabel, David; Walsh, Douglas S.

doi:10.4269/ajtmh.2011.10-0674

Cited by 52 publications

(66 citation statements)

References 39 publications

(50 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…A previously described SYBR green I-based assay (19,20) was used to test P. falciparum field isolates for in vitro susceptibility to doxycycline hyclate. Doxycycline was obtained from a commercial supplier (Sigma-Aldrich, Co., St. Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

Reduced In Vitro Doxycycline Susceptibility in Plasmodium falciparum Field Isolates from Kenya Is Associated with PfTetQ KYNNNN Sequence Polymorphism

Achieng

Ingasia

Juma

et al. 2014

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

Doxycycline is widely used for malaria prophylaxis by international travelers. However, there is limited information on doxycycline efficacy in Kenya, and genetic polymorphisms associated with reduced efficacy are not well defined. In vitro doxycycline susceptibility profiles for 96 Plasmodium falciparum field isolates from Kenya were determined. Genetic polymorphisms were assessed in P. falciparum metabolite drug transporter (Pfmdt) and P. falciparum GTPase tetQ (PftetQ) genes. Copy number variation of the gene and the number of KYNNNN amino acid motif repeats within the protein encoded by PftetQ were determined. Reduced in vitro susceptibility to doxycycline was defined by 50% inhibitory concentrations (IC 50 s) of >35,000 nM. The odds ratio (OR) of having 2 PfTetQ KYNNNN amino acid repeats in isolates with IC 50 s of >35,000 nM relative to those with IC 50 s of <35,000 nM is 15 (95% confidence interval [CI], 3.0 to 74.3; P value of <0.0002). Isolates with 1 copy of the Pfmdt gene had a median IC 50 of 6,971 nM, whereas those with a Pfmdt copy number of >1 had a median IC 50 of 9,912 nM (P ‫؍‬ 0.0245). Isolates with 1 copy of PftetQ had a median IC 50 of 6,370 nM, whereas isolates with a PftetQ copy number of >1 had a median IC 50 of 3,422 nM (P < 0.0007). Isolates with 2 PfTetQ KYNNNN motif repeats had a median IC 50 of 26,165 nM, whereas isolates with 3 PfTetQ KYNNNN repeats had a median IC 50 of 3,352 nM (P ‫؍‬ 0.0023). PfTetQ sequence polymorphism is associated with a reduced doxycycline susceptibility phenotype in Kenyan isolates and is a potential marker for susceptibility testing.

show abstract

Section: Methodsmentioning

confidence: 99%

Reduced In Vitro Doxycycline Susceptibility in Plasmodium falciparum Field Isolates from Kenya Is Associated with PfTetQ KYNNNN Sequence Polymorphism

Achieng

Ingasia

Juma

et al. 2014

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

show abstract

“…The technique has been utilized for in vitro and ex vivo profiling of field isolate susceptibility to antimalarials (12)(13)(14). However, there are conflicting published data on its performance in isolates with low parasitemia and high hematocrit levels (15)(16)(17).…”

mentioning

confidence: 99%

“…In our initial validation of the SYBR green assay in laboratory clones, after testing an extensive drug panel, including antibiotics and antifolates, and comparing these results to the [ 3 H]hypoxanthine assay, we obtained drug sensitivity profiles that were similar to those obtained by Smilkstein et al (8). In a follow-up study, we described the effect of assay components, including drugs and white blood cells, on the assay background signal, with the purpose of clarifying the applicability of the technique for ex vivo drug testing (12). Since then, the SYBR green assay has been used successfully to determine the 50% inhibitory concentrations (IC 50 s) of clinical isolates in comparison with the histidine-rich protein-2 (HRP2) enzyme-linked immunosorbent assay (ELISA) and the [ 3 H]hypoxanthine assay.…”

mentioning

confidence: 99%

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

Cheruiyot

Auschwitz

Lee

et al. 2016

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…57 Drugs, extracts and compounds were dissolved in 99.5% dimethylsulfoxide (DMSO) (Sigma-Aldrich) and diluted in complete Roswell Park Memorial Institute 1640 series of Cell Culture Media (RPMI 1640) enriched with human serum. The RPMI 1640 medium was prepared accordingly as described by Akala et al (2011). 58 Briefly, the basic culture medium was prepared from RPMI 1640 powder (10.4 g; Invitrogen, Inc. augmented with 2 g glucose (Sigma Inc.) and 5.95 g of HEPES (Sigma Inc.), dissolved to homogeneity in 1 L of de-ionized water and sterilized with a 0.2 µM filter.…”

Section: In Vitro Anti-plasmodial Assaymentioning

confidence: 99%

“…The RPMI 1640 medium was prepared accordingly as described by Akala et al (2011). 58 Briefly, the basic culture medium was prepared from RPMI 1640 powder (10.4 g; Invitrogen, Inc. augmented with 2 g glucose (Sigma Inc.) and 5.95 g of HEPES (Sigma Inc.), dissolved to homogeneity in 1 L of de-ionized water and sterilized with a 0.2 µM filter. Complete RPMI 1640 media, used for all parasite culture and drug dilutions, consisted of basic RPMI 1640 media with 10% (v/v) human ABO pooled plasma, 3.2% (v/v) sodium bicarbonate (Thermo Fisher Scientific Inc.) and 4 µg/mL hypoxanthine (Sigma Inc.).…”

Section: In Vitro Anti-plasmodial Assaymentioning

confidence: 99%

Antiplasmodial Activities of the Stem bark Extract and Compounds of Zanthoxylum gilletii (De wild) P.G. Waterman

Omosa¹,

Okemwa²

2017

View full text Add to dashboard Cite

Introduction: Multidrug resistance remains a major obstacle hindering successful antimalarial chemotherapy. In the current study, 50% methanol in dichloromethane extract and six compounds from the stem bark of Zanthoxylum gilletii were explored for their antiplasmodial potential against three strains of Plasmodium falciparum. Materials and Methods: The extract was obtained by cold percolation using 50 % MeOH in CH 2 Cl 2 and the antiplasmodial activities were assayed using a non-radioactive Malaria SYBR Green I assay to determine a concentration that inhibits growth of 50% of parasites in culture (IC 50 ). Results and Discussion: Chromatographic separation of the crude extract yielded six known compounds including: one lignan, sesamin (1), an alkamide, fagaramide (2), three benzo [c] phenanthridine alkaloids, 8-acetonyldihydrochelerythrine (3), dihydrochelerythine (4), norchelerythrine (5) and one pentacyclic triterpenoid, lupeol (6). The extract and sesamin (1) showed promising antiplasmodial activities against the chloroquine resistant (W2), chloroquine sensitive (D6) and 3D7 strains of P. falciparum, with IC 50 values of 2.52, 1.48 and 1.43 µg/mL and 5.4, 9.1 and 8.3 µM, respectively. To the best of our knowledge, this is the first report on antiplasmodial activities of the stem bark extract (50%

show abstract

Antimalarial Drug Sensitivity Profile of Western Kenya Plasmodium falciparum Field Isolates Determined by a SYBR Green I in vitro Assay and Molecular Analysis

Cited by 52 publications

References 39 publications

Reduced In Vitro Doxycycline Susceptibility in Plasmodium falciparum Field Isolates from Kenya Is Associated with PfTetQ KYNNNN Sequence Polymorphism

Reduced In Vitro Doxycycline Susceptibility in Plasmodium falciparum Field Isolates from Kenya Is Associated with PfTetQ KYNNNN Sequence Polymorphism

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

Antiplasmodial Activities of the Stem bark Extract and Compounds of Zanthoxylum gilletii (De wild) P.G. Waterman

Contact Info

Product

Resources

About