Search

Hemozoin, the heme detoxification end product in malaria parasites during their growth in the red blood cells (RBCs), serves as an important marker for diagnosis and treatment target of malaria disease. However, the current method for hemozoin-targeted drug screening mainly relies on in-vitro β-hematin inhibition assays, which may lead to false-positive events due to under-representation of the real hemozoin crystal. Quantitative in-situ imaging of hemozoin is highly desired for high-throughput screening of antimalarial drugs and for elucidating the mechanisms of antimalarial drugs. We present transient absorption (TA) imaging as a high-speed single-cell analysis platform with chemical selectivity to hemozoin. We first demonstrated that TA microscopy is able to identify β-hematin, the artificial form of hemozoin, from the RBCs. We further utilized time-resolved TA imaging to in situ discern hemozoin from malaria-infected RBCs with optimized imaging conditions. Finally, we quantitatively analyzed the hemozoin amount in RBCs at different infection stages by single-shot TA imaging. These results highlight the potential of TA imaging for efficient antimalarial drug screening and drug mechanism investigation.

Carrying out chemical analysis of antimalarials to detect low-quality medications before they reach a patient is a costly venture. Here, we show that a library of chemical color tests embedded on a paper card can presumptively identify formulations corresponding to very low quality antimalarial drugs. The presence or absence of chloroquine (CQ), doxycycline (DOX), quinine, sulfadoxine, pyrimethamine, and primaquine antimalarial medications, in addition to fillers used in low-quality pharmaceuticals, are indicated by patterns of colors that are generated on the test cards. Test card sensitivity for detection of these pure components ranges from 90% to 100% with no false positives in the absence of pharmaceutical. The color intensities from reactions characteristic of CQ or DOX allowed visual detection of formulations of these medications cut with 60% or 100% filler, although samples cut with 30% filler could not be reliably detected colorimetrically. However, the addition of unexpected fillers, even in 30% quantities, or substitute pharmaceuticals, could sometimes be detected by other color reactions on the test cards. Tests are simple and inexpensive enough to be carried out in clinics, pharmacies, and ports of entry and could provide a screening method to presumptively indicate very low quality medicines throughout the supply chain.

With the emergence of artesunate antimalarial counterfeiting in Southeast Asia and sub-Saharan Africa, we present the production of a rapid, inexpensive and simple colorimetric-based testing kit for the detection of counterfeit artesunate in order to preserve life and prevent the development of multi-drug resistant malaria. The kit works based on paper microfluidics which offer several advantages over conventional microfluidics, and has great potential to generate inexpensive, easy-to-use, rapid and disposable diagnostic devices. Here, we have developed a colorimetric assay that is specific to artesunate and turns yellow upon addition of the sample. The test can be done within minutes, and allows for a semi-quantitative analysis of the artesunate tablets by comparing the developed yellow color on the paper test to a color-coded key chart that comes with the kit. A more accurate and precise analysis is done by utilizing a color analyzer on an iPhone camera that measures the color intensity of the developed color on the paper chip. A digital image of the chip was taken and analyzed by measuring the average gray intensity of the color developed on the paper circle. A plot of the artesunate concentration versus the average gray scale intensity was generated. Results show that the intensity of the yellow color developed on the paper test was consistent and proportional to the amount of artesunate present in the sample. With artesunate concentrations ranging from 0.0 to 20 mg/mL, a linear calibration plot was obtained with a detection limit of 0.98 mg/mL.

A method for the determination of sulfadoxine and sulfamethoxazole in capillary blood on sampling paper has been developed and validated. The method is straightforward with minimal sample preparation, and is suitable for rural settings. Separation of sulfadoxine, sulfamethoxazole and internal standard was performed using a Purospher STAR RP-18 endcapped LC column (150x4.6mm) with a mobile phase consisting of acetonitrile:sodium acetate buffer pH 5.2, I=0.1 (1:2, v/v). For sulfadoxine, the within-day precision was 5.3% at 15micromol/l and 3.7% at 600micromol/l, while for sulfamethoxazole it was 5.7% at 15micromol/l and 3.8% at 600micromol/l. The lower limit of quantification was determined to 5micromol/l and precision was 5.5% and 5.0% for sulfadoxine and sulfamethoxazole, respectively.

Malaria is a preventable disease, which is under notified in the U.K. This study sought to evaluate the current surveillance arrangements in Northern Ireland (NI), describe the epidemiology of malaria and make appropriate recommendations. A case was defined as a resident or visitor to NI with laboratory confirmed malaria, diagnosed by the NI haematology laboratories and/or the Malaria Reference Laboratory (MRL) from 1998-2003. Laboratory data were compared with notifications and hospital admission data. One hundred and fourteen laboratory cases were identified compared with 63 notifications received by the regional surveillance centre. Six cases were associated with two episodes of malaria reflecting recurrence and or reinfection. P falciparum was the most common infection with two fatalities reported; this was particularly associated with travel to West Africa. Most cases were associated with short visits to malarious areas. Thirty-three percent of all cases did not take prophylaxis and, of those that did, approximately half were taking a prophylactic regime appropriate to the region visited. This study highlights the need for improved surveillance of malaria in order to capture risk factors and other relevant information to inform public and professional education. This would facilitate increasing local awareness, enhancing prescription of and compliance with appropriate chemoprophylaxis and enabling early diagnosis and treatment of malaria.

A cost-effective HPLC method for determination of pyrimethamine (PYR) in human whole blood samples dried on filter paper (Whatman) is reported. Trimethoprim (TMP) was used as an internal standard. Whole blood spiked with PYR was transferred (100 microl) onto filter paper and dried at room temperature. Capillary blood samples (100 microl) after ingestion of three tablets of sulfadoxine-pyrimethamine (SP) by one subject were also tested. PYR and an internal standard (IS) TMP were extracted into di-isopropyl ether as bases and then re-extracted with 150 microl mobile phase. A C-18 column was used and the mobile phase consisted of phosphate buffer (0.05 M, pH 5):acetonitrile:concentrated perchloric acid (750:300:2.5, v/v/v). The absorbances of PYR and IS were monitored at 270 nm. The limit of quantification was 40 ng/ml. The within- and between-assay coefficient of variations were

Where malaria surveillance and health care is inadequate, iron supplements given without food can increase the severity of malarial infections. The likely explanation is that the rate of iron influx into the plasma from high-dose oral supplements exceeds the rate of iron binding to transferrin and a quantity of non-transferrin-bound iron (NTBI) is formed. It is proposed that NTBI increases the intensity of malarial infections by increasing the sequestration of malaria-infected red cells in the capillaries of the brain and intestine, causing more cerebral malaria and further increasing the permeability of the intestinal barrier to the passage of pathogens. Bacteremia is frequently reported in children with severe malaria. At the same time, high iron doses stimulate the growth of pathogenic bacteria in the stool, further increasing the potential for bacteremia. The normal immune response to malaria, as well as other infections and inflammatory disorders, is to prevent further microbial growth by stimulating hepcidin synthesis and preventing the passage of iron into the plasma. Iron absorption is decreased and the efficacy of the iron interventions would be expected to be lower in the presence of infections.

A bioanalytical method for the determination of lumefantrine in 100 microl blood applied onto sampling paper, by solid-phase extraction and liquid chromatography, has been developed and validated. Whatman 31 ET Chr sampling paper was pre-treated with 0.75 M tartaric acid before sampling capillary blood to enable a high recovery of lumefantrine. Lumefantrine was extracted from the sampling paper, then further purified using solid-phase extraction and finally quantified with HPLC. The between-day variation was below 10% over the range 0.4-25 microM. The lower limit of quantification was 0.25 microM in 100 microl capillary blood. No decrease in lumefantrine concentration in dried blood spot is seen after 4 months storage at 22 degrees C. The method was also evaluated in field samples from patients in Tanzania after treatment with lumefantrine/artemether. Lumefantrine could be estimated accurately enough to assess bioavailability and treatment compliance on day 7 (i.e. 4 days after the last dose) after a standard regimen with the lumefantrine/artemether combination.

An improved method is presented for the determination of proguanil, cycloguanil and 4-chlorophenylbiguanide in 100-μl capillary blood samples applied to sampling paper. This method also utilises a solid-phase extraction technique and high-performance liquid chromatography. Different kinds of sampling paper, such as ion-exchange and cellulose sampling paper were tested. The best elution recovery (70–80%) was obtained after treatment of cellulose sampling paper with a quaternary ammonium compound. The limit of determination was 50 nmol/l for cycloguanil and 4-chlorophenylbiguanide and 125 nmol/l for proguanil using 100 μl capillary blood. The stability of the analytes and elution performance from sampling paper was validated at different temperature and storage time. Venous blood and capillary blood concentrations of proguanil and metabolites were found to be similar.

Mefloquine is determined in 100-microliter samples of whole blood, plasma and capillary blood collected on filter paper by gas chromatography with electron-capture detection after derivatization with phosgene. Sample preparation for whole blood and plasma involves a protein precipitation step that uses a combination of zinc and acetonitrile, followed by simultaneous extraction with methylene chloride and derivatization with phosgene at pH 9.50. Filter paper spots are immersed for 12-24 h in 0.1 M hydrochloric acid, followed by simultaneous extraction with methyl tert.-butyl ether and derivatization. After evaporation of the organic phase and reconstitution with ethyl acetate, 1 microliter of the extract is injected into a megabore capillary column. Because of the high sensitivity of the method, mefloquine concentrations down to 25 nmol/l (9.5 micrograms/l) are determined in 100-microliters samples with a relative standard deviation of 12% at the 25 nmol/l level. Excellent precision was obtained over the range of concentrations tested, 0.10-3 mumol/l (45-1100 micrograms/l), in both plasma and whole blood and from filter-paper-collected capillary blood. The day-to-day relative standard deviation in plasma at the therapeutic level (1-3 mumol/l) was 4.5% (n = 8).