Flow cytometry is a powerful tool for measuring parasitemias in murine malaria models used to test new antimalarials. Measurement of the emission of the nonpermeable nucleic acid dye YOYO-1 (at 530 and 585 nm after excitation at 488 nm) allowed the unambiguous detection of low parasitemias (!0.01%) but required prolonged fixation and permeabilization of the sample. Thus, we tested whether this issue could be overcome by use of the cell-permeant dye SYTO-16 with this same bidimensional method. Blood samples from CD1 mice infected with Plasmodium yoelii, Plasmodium vinckei, or Plasmodium chabaudi or from NOD scidb2m-/-engrafted with human erythrocytes and infected with P. falciparum were stained with SYTO-16 in the presence or absence of TER-119 mAb (for engrafted mice) in 96-well plate format and acquired in Trucount TM tubes. Bidimensional analysis with SYTO-16 was quantitatively equivalent to YOYO-1. Moreover, by combining SYTO-16 with the use of TER-119-PE antimouse erythrocyte mAb and Trucount tubes, the measurement of the concentration of P. falciparuminfected erythrocytes over a range of five orders of magnitude was achieved. Bidimensional analysis using SYTO-16 can be used to accurately measure the concentration of Plasmodium spp.-infected erythrocytes in mice without complex sample preparation. MALARIA is caused by the erythrocytic stages of protozoa of the genus Plasmodium, which colonize and destroy host's erythrocytes (1). To counter this disease, murine models of malaria are essential tools for research (2), particularly for drug discovery (3). In addition to the standard rodent experimental systems, different murine models of P. falciparum malaria are currently available (4-6). These are of special interest for drug discovery because, with the exception of human subjects, these are the only experimental systems available that allow the evaluation in vivo of the real human pathogen growing inside human erythrocytes (hE) previously engrafted into immunodeficient mice. Not surprisingly, the peripheral blood of these chimeric mice [humanized mice (HM)] is a complex mixture of murine erythrocytes (mE) and hE, in which the hematological effects of massive transfusions of hE and their elimination from peripheral blood may have important effects. Hence, the specific and quantitative measurement of different erythrocytic subpopulations is crucial in HM models, particularly when these models are used to establish the relationship between the amount of an antimalarial drug in blood and the effect on parasitemia through experimental pharmacokinetic and pharmacodynamic studies (PK/PD). In this kind of

Genz-644442 became the focus of medicinal chemistry optimization; 321 analogs were synthesized and were tested for in vitro potency against P. falciparum and for in vitro absorption, distribution, metabolism, and excretion (ADME) properties. This yielded compounds with IC 50 s of approximately 30 nM. The lead compound, Genz-668764, has been characterized in more detail. It is a single enantiomer with IC 50 s of 28 to 65 nM against P. falciparum in vitro. In the 4-day P. berghei model, when it was dosed at 100 mg/kg of body weight/day, no parasites were detected on day 4 postinfection. However, parasites recrudesced by day 9. Dosing at 200 mg/kg/day twice a day resulted in cures of 3/5 animals. The compound had comparable activity against P. falciparum blood stages in a humanengrafted NOD-scid mouse model. Genz-668764 had a terminal half-life of 2.8 h and plasma trough levels of 41 ng/ml when it was dosed twice a day orally at 55 mg/kg/day. Seven-day rat safety studies showed a no-observableadverse-effect level (NOAEL) at 200 mg/kg/day; the compound was not mutagenic in Ames tests, did not inhibit the hERG channel, and did not have potent activity against a broad panel of receptors and enzymes. Employing allometric scaling and using in vitro ADME data, the predicted human minimum efficacious dose of Genz-668764 in a 3-day once-daily dosing regimen was 421 mg/day/70 kg, which would maintain plasma trough levels above the IC 90 against P. falciparum for at least 96 h after the last dose. The predicted human therapeutic index was approximately 3, on the basis of the exposure in rats at the NOAEL. We were unable to select for parasites with >2-fold decreased sensitivity to the parent compound, Genz-644442, over 270 days of in vitro culture under drug pressure. These characteristics make Genz-668764 a good candidate for preclinical development.Malaria continues to be a major global health burden, endemic in 87 countries with 2.5 billion people at risk (11). Widespread resistance to current antimalarials such as chloroquine (29, 40), atovaquone (33), pyrimethamine (39), and sulfadoxine (37) and, more recently, reduced efficacy of artemisinin derivatives (7, 38) emphasize the urgent need for new antimalarial drugs. At least in part because the majority of people affected live in the poorest parts of the world, the response of the pharmaceutical industry has been sparse or irregular. New collaborations combining the expertise of academia, industry, and public-private partnerships have been suggested to overcome these obstacles, and it is in that spirit

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Glucose 6 phosphate dehydrogenase deficiency. A case seriesResumen Describimos las características clínicas y de laboratorio de 50 individuos con deficiencia de glucosa-6-fosfato deshidrogenasa (D-G6PD). La D-G6PD representó el 1,1% de los diagnósti-cos realizados. Se detectó la coexistencia de D-G6PD con otra eritropatía: G6PD/Hb S en 2 pacientes y G6PD/esferocitosis congénita en 1 paciente. Todos los varones (100%) presentaron una prueba de Brewer (PB) positiva, pero sólo el 56% de las mujeres la presentaron. La actividad enzimática media (AEM) de los varones fue de 0,85 ± 0,52 U/g Hb. La AEM de las mujeres con PB positiva fue de 3,82 ± 1,26 U/g Hb y fue de 5,65 ± 2,84 U/g Hb en las mujeres con PB negativa. Todos los individuos recibieron asesoramiento genético y la lista de fármacos y alimentos con efecto oxidante. Resaltamos la importancia de incluir una prueba de pesquisa en el estudio de las anemias, para detectar individuos asintomáticos y la coexistencia con otras eritropatías. Palabras clave: enzimopatía, G6PD, anemia hemolítica, ictericia. AbstRActWe describe the laboratory and clinical characteristics of 50 patients with glucose 6 phosphate dehydrogenase deficiency (G6PD). G6PD deficiency represented 1.1% of all the diagnosis made. Coexistence of G6PD with other erythropathy was detected as follow: G6PG/HbS 2 patients and G6PG/hereditary spherocytosis 1 patient. A positive Brewer's test was found in 100% of males but in only 56% of women. Males had a mean enzymatic activity (MEA) of 0.85 ± 0.52 U/g Hb. Women, with positive Brewer's test, showed a MEA of 3.82 ± 1.26 U/g Hb, while the MEA of women with negative Brewer's test was 5.65 ± 2.84 U/g Hb. Genetic counseling and the list of food and drugs potentially harmful was given to all patients. The inclusion of simple screening tests, such as Brewer's test, in the study of anemia, enables us to detect asymptomatic males and carriers in whom this enzymopathy was co-inherited with another erythropathy.

BackgroundQuantitative real-time PCR (qPCR) is now commonly used as a method to confirm diagnosis of malaria and to differentiate recrudescence from re-infection, especially in clinical trials and in reference laboratories where precise quantification is critical. Although anti-malarial drug discovery is based on in vivo murine efficacy models, use of molecular analysis has been limited. The aim of this study was to develop qPCR as a valid methodology to support pre-clinical anti-malarial models by using filter papers to maintain material for qPCR and to compare this with traditional methods.MethodsFTA technology (Whatman) is a rapid and safe method for extracting nucleic acids from blood. Peripheral blood samples from mice infected with Plasmodium berghei, P. yoelii, or P. falciparum were kept as frozen samples or as spots on FTA cards. The extracted genetic material from both types of samples was assessed for quantification by qPCR using sets of specific primers specifically designed for Plasmodium 18S rRNA, LDH, and CytB genes.ResultsThe optimal conditions for nucleic acid extraction from FTA cards and qPCR amplification were set up, and were confirmed to be suitable for parasite quantification using DNA as template after storage at room temperature for as long as 26 months in the case of P. berghei samples and 52 months for P. falciparum and P. yoelii. The quality of DNA extracted from the FTA cards for gene sequencing and microsatellite amplification was also assessed.ConclusionsThis is the first study to report the suitability of FTA cards and qPCR assay to quantify parasite load in samples from in vivo efficacy models to support the drug discovery process.