Devices for rapid diagnosis of Malaria: evaluation of prototype assays that detect Plasmodium falciparum histidine-rich protein 2 and a Plasmodium vivax-specific antigen

The ParaSight F test was developed as a pioneer industry effort in the large-scale, process-controlled production of a device for the rapid diagnosis of malaria. This device performed well in field settings but was limited to the detection of a single malaria species, Plasmodium falciparum. The ParaSight F+V assay advanced upon the ParaSight F test format by incorporating a monoclonal antibody directed against a proprietary Plasmodium vivax-specific antigen, in addition to the antibody directed against P. falciparum histidine-rich protein 2, which was used in the ParaSight F assay. The modified assay was developed to add the capability to detect P. falciparum and P. vivax in a single-test-strip format. The present study evaluated three distinct ParaSight F+V prototypes with samples from symptomatic patients in regions of Thailand and Peru where malaria is endemic. Over a 2-year enrollment period (1998 and 1999), a total of 4,894 patients consented to participation in the study. Compared with the results for duplicate microscopic examinations of Giemsa-stained blood smears as the reference diagnostic standard, each successive prototype showed substantial improvement in performance. The final ParaSight F+V prototype, evaluated in 1999, had an overall sensitivity for detection of asexual P. falciparum parasites of 98%. The sensitivity of the device was 100% for P. falciparum densities of >500 parasites/ micro l, with a sensitivity of 83% for parasite densities of 5,000/ micro l, 92% for parasite densities of 1,001 to 5,000/ micro l, 94% for parasite densities of 501 to 1,000/ micro l, and 55% for parasite densities of 1 to 500/ micro l. The specificity for the exclusion of P. vivax was 87%. The areas under the receiver operating characteristic curves for the diagnostic performance of the assay for the detection of P. falciparum and P. vivax were 0.8907 and 0.8522, respectively. These findings indicate that assays for rapid diagnosis have the potential to enhance diagnostic capabilities in those instances in which skilled microscopy is not readily available.     

Comparison of the in-vitro activity of amodiaquine and its main metabolite, monodesethyl-amodiaquine, in Plasmodium falciparum

After its rehabilitation for therapeutic use in uncomplicated falciparum malaria, there is renewed interest in amodiaquine. After oral administration, the drug undergoes rapid metabolism to monodesethyl-amodiaquine, and in patients with normal hepatic function the parent drug usually becomes undetectable within a few hours. The main antimalarial activity is therefore mainly due to the metabolite. In a comparative study in northwestern Thailand, 21 fresh isolates of Plasmodium falciparum were tested, in parallel, for their in-vitro sensitivity to both compounds, using the WHO micro-test Mark II, measuring the inhibition of schizont maturation. The geometric mean cut-off concentrations of schizont maturation were 1826 nM (related to blood) for amodiaquine, and 1654 nM for monodesethyl-amodiaquine. The log-probit regressions for both compounds showed good fits to the data points. The EC50 values were 331 nM and 291 nM, and the EC90 values 1337 nM and 993 nM for amodiaquine and monodesethyl-amodiaquine, respectively. Differences between regression slopes and effective concentrations were well below statistical significance. Both compounds showed highly significant activity correlation. These findings suggest that the sensitivity of Plasmodium falciparum to amodiaquine closely reflects its sensitivity to monodesethyl-amodiaquine.     

Synergism of desbutyl-benflumetol and retinol against Plasmodium falciparum in vitro

This in vitro study was conducted to assess the blood schizontocidal activity of desbutyl-benflumetol (DBB), a new benzindene derivative, retinol and a combination of both compounds. The tests were carried out according to the methodology of the WHO standard test Mark II, measuring the drug-dependent inhibition of schizont maturation, and using 43 fresh isolates of Plasmodium falciparum from northwestern Thailand, an area with established multidrug-resistance. DBB and retinol showed a mean 50% effective concentration (EC-50) of 3.73 nM and 466.86 nM and 90% effective concentration (EC-90) of 19.83 nM and 5531.69 nM respectively. The combination of DBB and 3.50 muM retinol showed strong inhibition of schizont maturation, with an EC-90 for DBB of 0.67 nM. At the therapeutically relevant EC-99, the combination was about 10 times more effective than expected, suggesting that retinol potentiated the effect of DBB. A concentration of 3.50 muM retinol corresponds to the 95th percentile of the physiological serum levels. It is well known that retinol levels are significantly decreased in acute falciparum malaria. Supplementation with retinol during malaria treatment may improve the therapeutic results of blood schizontocides of the fluorene class.     

Comparative study on the in vitro activity of lumefantrine and desbutyl-benflumetol in fresh isolates of Plasmodium vivax from Thailand

The occurrence of chloroquine resistance in Plasmodium vivax underlines the need for monitoring the drug response of this important malaria parasite and for the evaluation of alternative therapeutic agents. In-vitro methods facilitate these tasks. This investigation employed a recently developed in-vitro micro-technique and validated it for lumefantrine and desbutyl-benflumetol, a compound that was initially considered a metabolite of lumefantrine. The studies were conducted in 2001 at Mae Sot, a town situated in northwestern Thailand near the border to Myanmar. Parallel in-vitro tests with lumefantrine and desbutyl-benflumetol were carried out with 53 fresh isolates of P. vivax. For both compounds, the parasite showed a homogenous, log-normal inhibition pattern with nearly parallel log-probit regressions. The geometric mean drug concentrations effecting complete growth inhibition were 2361 nM for lumefantrine and 187 nM for desbutyl-benflumetol. With p=3.264 x 10(-18) the difference was highly significant. The EC50 and EC90 values for lumefantrine, 17.6 nM and 448.5 nM, respectively, were much higher as compared to those determined for desbutyl-benflumetol, with 1.5 nM and 39.7 nM. This difference expressed itself in a highly significant Power Ratio (PR) of 11.0. The activity of desbutyl-benflumetol in P. vivax exceeds that of lumefantrine by one order of magnitude, suggesting a high, hitherto unexploited therapeutic potential of desbutyl-benflumetol.     

Spatio-temporal distribution of Plasmodium falciparum and p. Vivax malaria in Thailand

Malaria incidence data at the district level from 1997 to 2002 and total malaria case data from 1965 to 2002 in Thailand were analyzed to determine the spatial and temporal dynamics of Plasmodium falciparum and P. vivax malaria incidence. Over the 37-year period, there was a 35-fold reduction in the incidence rates of P. falciparum malaria (11.86% in 1965 versus 0.34% in 2002) and a 7-fold reduction in P. vivax malaria (2.89% in 1965 versus 0.40% in 2002). The incidence ratio of P. falciparum to P. vivax malaria was reduced from 4.1 to 0.8 during this period. Malaria incidence rate exhibited the most rapid reduction between 1975 and 1985, coinciding with the introduction of a combination of antifolate drugs (sulfadoxine-pyrimethamine). The distribution maps of P. falciparum and P. vivax malaria incidence rates indicated a high spatial heterogeneity. The Thailand-Myanmar and Thailand-Cambodia border areas, where migration of foreign workers was pronounced, had the highest incidence rates for P. falciparum, P. vivax, and mixed-species infections. Transition probability analysis based on the malaria incidence rate among Thai residents indicated that there was an overall trend of decrease in the number of malaria cases and the number of high incidence districts between 1997 and 2002. High spatial variation in malaria incidence and local human migration patterns suggest that malaria control measures need to be adjusted according to local environmental and demographic settings.     

A Comparison of 3 Quantitative Radiographic Measurement Methods for Root Development Measurement in Regenerative Endodontic Procedures

Introduction

The outcomes of an immature tooth with necrotic pulp treated with regenerative endodontic procedures (REPs) were assessed clinically and radiographically. Root maturation is an important outcome of REPs, and several radiographic measurement methods have been used to measure this. The aim of this study was to compare radiographic measurement methods, measuring root maturation in immature teeth with necrotic pulp treated with REPs.

An in vitro system for assessing the sensitivity of Plasmodium vivax to chloroquine

Following earlier observations on short-term culture of Plasmodium vivax, an in vitro test system has been developed for assessing the parasite's sensitivity to chloroquine. Fresh isolates with predominantly young trophozoites are diluted 1:19 with a (v/v=1/1) mixture of RPMI 1640 and Waymouth medium. The blood-medium mixture (BMM) is inoculated into the predosed microtitre plates before incubation in a candle jar. Incubation for 30 or 42 h yielded the best results. Incubation for 18 or 24 h was generally insufficient for an adequate development of the parasites. The reading of the test is based on stage-specific differential counts in the Giemsa-stained pre-incubation and post-incubation thick films, the evaluation on log-probit analysis of drug-related inhibition of parasite development. The test system has been evaluated on 200 fresh P. vivax isolates in an area with satisfactory clinical-parasitological response to chloroquine. At 30 or 42 h incubation 121 isolates (61.5%) showed adequate control growth and yielded valid sensitivity tests. Complete inhibition of parasite development occurred within the concentration range of 40-1280 nM. The mean EC50 for 30 h of incubation was 50.3 nM, as compared to 49.7 nM with 42 h of incubation. The geometric mean cut-off concentration of parasite development was 488 nM with 30 h of incubation as against 470 nM with 42 h of incubation.