scholarly journals Determinants of In Vitro Drug Susceptibility Testing of Plasmodium vivax

2008 ◽  
Vol 52 (3) ◽  
pp. 1040-1045 ◽  
Author(s):  
B. Russell ◽  
F. Chalfein ◽  
B. Prasetyorini ◽  
E. Kenangalem ◽  
K. Piera ◽  
...  
Keyword(s):  
Plasmodium Vivax ◽  
Specific Activity ◽  
Geometric Mean ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Trophozoite Stage ◽  
Initial Stage ◽  
Two Factors

ABSTRACT In Papua, Indonesia, the antimalarial susceptibility of Plasmodium vivax (n = 216) and P. falciparum (n = 277) was assessed using a modified schizont maturation assay for chloroquine, amodiaquine, artesunate, lumefantrine, mefloquine, and piperaquine. The most effective antimalarial against P. vivax and P. falciparum was artesunate, with geometric mean 50% inhibitory concentrations (IC50s) (95% confidence intervals [CI]) of 1.31 nM (1.07 to 1.59) and 0.64 nM (0.53 to 0.79), respectively. In contrast, the geometric mean chloroquine IC50 for P. vivax was 295 nM (227 to 384) compared to only 47.4 nM (42.2 to 53.3) for P. falciparum. Two factors were found to significantly influence the in vitro drug response of P. vivax: the initial stage of the parasite and the duration of the assay. Isolates of P. vivax initially at the trophozoite stage had significantly higher chloroquine IC50s (478 nM [95% CI, 316 to 722]) than those initially at the ring stage (84.7 nM [95% CI, 45.7 to 157]; P < 0.001). Synchronous isolates of P. vivax and P. falciparum which reached the target of 40% schizonts in the control wells within 30 h had significantly higher geometric mean chloroquine IC50s (435 nM [95% CI, 169 to 1,118] and 55.9 nM [95% CI, 48 to 64.9], respectively) than isolates that took more than 30 h (39.9 nM [14.6 to 110.4] and 36.9 nM [31.2 to 43.7]; P < 0.005). The results demonstrate the marked stage-specific activity of chloroquine with P. vivax and suggest that susceptibility to chloroquine may be associated with variable growth rates. These findings have important implications for the phenotypic and downstream genetic characterization of P. vivax.

scholarly journals In VivoandIn VitroEfficacy of Chloroquine againstPlasmodium malariaeandP. ovalein Papua, Indonesia

2010 ◽  
Vol 55 (1) ◽  
pp. 197-202 ◽  
Author(s):  
H. Siswantoro ◽  
B. Russell ◽  
A. Ratcliff ◽  
B. Prasetyorini ◽  
F. Chalfein ◽  
...  
Keyword(s):  
Parasite Clearance ◽  
Plasmodium Malariae ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Chloroquine Resistance ◽  
Ring Stage ◽  
Trophozoite Stage ◽  
Nm Range

ABSTRACTReports of potential drug-resistant strains ofPlasmodium malariaein western Indonesia raise concerns that chloroquine resistance may be emerging inP. malariaeandP. ovale. In order to assess this,in vivoandin vitroefficacy studies were conducted in patients with monoinfection in Papua, Indonesia. Consecutive patients with uncomplicated malaria due toP. ovaleorP. malariaewere enrolled in a prospective clinical trial, provided with supervised chloroquine treatment, and followed for 28 days. Blood from patients withP. malariaeorP. ovaleparasitemia greater than 1,000 per microliter underwentin vitroantimalarial drug susceptibility testing using a modified schizont maturation assay. Of the 57 evaluable patients in the clinical study (P. malariae,n= 46;P. ovale,n= 11), none had recurrence with the same species during follow-up. The mean parasite reduction ratio at 48 h was 86 (95% confidence interval [CI], 57 to 114) forP. malariaeand 150 (95% CI, 54 to 245) forP. ovale(P= 0.18). One patient infected withP. malariae, with 93% of parasites at the trophozoite stage, was still parasitemic on day 4.In vitrodrug susceptibility assays were carried out successfully for 40 isolates (34 infected withP. malariaeand 6 withP. ovale). TheP. malariaeinfections at trophozoite stages had significantly higher chloroquine 50% effective concentrations (EC50s) (median, 127.9 nM [range, 7.9 to 2,980]) than those initially exposed at the ring stage (median, 14.0 nM [range, 3.5 to 27.0];P= 0.01). The EC50for chloroquine inP. ovalewas also higher in an isolate initially at the trophozoite stage (23.2 nM) than in the three isolates predominantly at ring stage (7.8 nM). Chloroquine retains adequate efficacy againstP. ovaleandP. malariae, but its marked stage specificity of action may account for reports of delayed parasite clearance times.

Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

1991 ◽  
Vol 66 (04) ◽  
pp. 453-458 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Specific Activity ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Clinical Importance ◽  
Potential Method ◽  
Quantitative Expression ◽  
Increased Risk ◽  
Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

scholarly journals Impact of the first-line treatment shift from dihydroartemisinin/piperaquine to artesunate/mefloquine on Plasmodium vivax drug susceptibility in Cambodia

2020 ◽  
Vol 75 (7) ◽  
pp. 1766-1771 ◽  
Author(s):  
Camille Roesch ◽  
Mélissa Mairet-Khedim ◽  
Saorin Kim ◽  
Dysoley Lek ◽  
Jean Popovici ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Vivax ◽  
Drug Susceptibility ◽  
Single Mutation ◽  
First Line ◽  
The Past ◽  
Drug Assays ◽  
Number Variation ◽  
In Vitro Response

Abstract Background Cambodia is the epicentre of the emergence of Plasmodium falciparum drug resistance. Much less is known regarding the drug susceptibility of the co-endemic Plasmodium vivax. Only in vitro drug assays can determine the parasite’s intrinsic susceptibility, but these are challenging to implement for P. vivax and rarely performed. Objectives To evaluate the evolution of Cambodian P. vivax susceptibility to antimalarial drugs and determine their association with putative markers of drug resistance. Methods In vitro response to three drugs used in the past decade in Cambodia was measured for 52 clinical isolates from Eastern Cambodia collected between 2015 and 2018 and the sequence and copy number variation of their pvmdr1 and pvcrt genes were analysed. pvmdr1 polymorphism was also determined for an additional 250 isolates collected in Eastern Cambodia between 2014 and 2019. Results Among the 52 cryopreserved isolates tested, all were susceptible to the three drugs, with overall median IC50s of 16.1 nM (IQR 11.4–22.3) chloroquine, 3.4 nM (IQR 2.1–5.0) mefloquine and 4.6 nM (IQR 2.7–7.0) piperaquine. A significant increase in chloroquine and piperaquine susceptibility was observed between 2015 and 2018, unrelated to polymorphisms in pvcrt and pvmdr1. Susceptibility to mefloquine was significantly lower in parasites with a single mutation in pvmdr1 compared with isolates with multiple mutations. The proportion of parasites with this single mutation genotype increased between 2014 and 2019. Conclusions P. vivax with decreased susceptibility to mefloquine is associated with the introduction of mefloquine-based treatment during 2017–18.

scholarly journals Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2

2011 ◽  
Vol 77 (9) ◽  
pp. 2926-2933 ◽  
Author(s):  
Kesaven Bhubalan ◽  
Jo-Ann Chuah ◽  
Fumi Shozui ◽  
Christopher J. Brigham ◽  
Seiichi Taguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Weight Percent ◽  
Pha Synthase ◽  
Content Type ◽  
Highly Active ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase

ABSTRACTThe synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolatedChromobacteriumsp. USM2 (PhaCCs). PhaCCsshowed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. Anin vitroassay of recombinant PhaCCsexpressed inEscherichia colishowed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strainC. necator(307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCswas 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC fromC. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation inEscherichia coliexpressing PhaCCsof up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaCCsis a naturally occurring, highly active PHA synthase with superior polymerizing ability.

scholarly journals A chemiluminescent protease probe for rapid, sensitive, and inexpensive detection of live Mycobacterium tuberculosis

2020 ◽  
Author(s):  
Brett M. Babin ◽  
Gabriela Fernandez-Cuervo ◽  
Jessica Sheng ◽  
Ori Green ◽  
Alvaro A. Ordonez ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Signal To Noise Ratio ◽  
Point Of Care ◽  
High Sensitivity ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
High Signal ◽  
Resource Limited ◽  
Tb Diagnostics

AbstractTuberculosis (TB) is a top-ten cause of death worldwide. Successful treatment is often limited by insufficient diagnostic capabilities, especially at the point of care in low-resource settings. The ideal diagnostic must be fast, cheap, and require minimal clinical resources while providing high sensitivity, selectivity, and the ability to differentiate live from dead bacteria. We describe here the development of a Fast, Luminescent, and Affordable Sensor of Hip1 (FLASH) for the diagnosis and monitoring of drug sensitivity of Mycobacterium tuberculosis (Mtb). FLASH is a selective chemiluminescent substrate for the Mtb protease Hip1 that when processed, produces visible light that can be measured with a high signal to noise ratio using inexpensive sensors. FLASH is sensitive to fmol of recombinant Hip1 enzyme in vitro and can detect as few as thousands of Mtb cells in culture or in human sputum samples within minutes. The probe is highly selective for Mtb compared to other non-tuberculous mycobacteria and can distinguish live from dead cells. Importantly, FLASH can be used to measure antibiotic killing of Mtb in culture with greatly accelerated timelines compared to traditional protocols. Overall, FLASH has the potential to enhance both TB diagnostics and drug resistance monitoring in resource-limited settings.One Sentence SummaryA luminescent probe enables sensitive detection of Mycobacterium tuberculosis for diagnostics, treatment monitoring, and drug susceptibility testing.

Novel application of agarose in cultivating microorganisms in the stomach and rapid drug susceptibility testing of Helicobacter pylori

2021 ◽  
Vol 11 (6) ◽  
pp. 880-887
Author(s):  
Qijuan Sun ◽  
Chengbo Li ◽  
Xiaona Xu ◽  
Haitao Zhao ◽  
Chenguang Liu
Keyword(s):  
Helicobacter Pylori ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
The Novel ◽  
Whole Process ◽  
Promising Tool ◽  
Growth Assay ◽  
Early Exploration

Agarose is a promising tool for encapsulating areas as a kind of neutral polysaccharide. The purpose of this work is to expand the application of agarose. In this work, agarose microparticles for encapsulating microorganisms were introduced to the stomach through a novel water-in-water (w/w) emulsification method. Sequencing techniques were performed for the identification and characterization of bacteria, and drug-susceptibility testing of Helicobacter pylori through gel microdroplets growth assay and traditional Oxford cup method was conducted. Results indicated the presence of three phyla, eight genera, and more than 30 species in the samples. The correlation values of the traditional Oxford cup and GMD methods were 87.5% and 90%, respectively. The proposed encapsulation technology as efficient substitution for traditional Oxford cup method promised to be applicable for the isolation and cultivation of gastric flora. Compared to other methods, this new method showed advantages when mainly due to time simplicity of the whole process. The direct drug susceptibility test based on the novel encapsulation technology is a promising tool for the rational and flexible use of drugs in clinical practice. Furthermore, this work was an early exploration for the combination of encapsulation technology and agarose.

scholarly journals Pyrazinamide Is Active against Mycobacterium tuberculosis Cultures at Neutral pH and Low Temperature

2016 ◽  
Vol 60 (8) ◽  
pp. 4956-4960 ◽  
Author(s):  
Alice L. den Hertog ◽  
Sandra Menting ◽  
Richard Pfeltz ◽  
Matthew Warns ◽  
Salman H. Siddiqi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Low Temperature ◽  
Susceptibility Testing ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Acidic Ph ◽  
Content Type ◽  
The Past ◽  
Neutral Ph

ABSTRACTFor the past decades, an acidic pH has been used to renderMycobacterium tuberculosissusceptible to pyrazinamide forin vitrotesting. Here, we show that at the standard breakpoint concentration and reduced culture temperatures, pyrazinamide (PZA) is active against tuberculosis (TB) at neutral pH. This finding should help unravel the mechanism of action of PZA and allow drug susceptibility testing (DST) methods to be optimized.

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