An automatic vision-based malaria diagnosis system.

Malaria is a worldwide health problem with 225 million infections each year. A fast and easy-to-use method, with high performance is required to differentiate malaria from non-malarial fevers. Manual examination of blood smears is currently the gold standard, but it is time-consuming, labour-intensive, requires skilled microscopists and the sensitivity of the method depends heavily on the skills of the microscopist. We propose an easy-to-use, quantitative cartridge-scanner system for vision-based malaria diagnosis, focusing on low malaria parasite densities. We have used special finger-prick cartridges filled with acridine orange to obtain a thin blood film and a dedicated scanner to image the cartridge. Using supervised learning, we have built a Plasmodium falciparum detector. A two-step approach was used to first segment potentially interesting areas, which are then analysed in more detail. The performance of the detector was validated using 5,420 manually annotated parasite images from malaria parasite culture in medium, as well as using 40 cartridges of 11,780 images containing healthy blood. From finger prick to result, the prototype cartridge-scanner system gave a quantitative diagnosis in 16 min, of which only 1 min required manual interaction of basic operations. It does not require a wet lab or a skilled operator and provides parasite images for manual review and quality control. In healthy samples, the image analysis part of the system achieved an overall specificity of 99.999978% at the level of (infected) red blood cells, resulting in at most seven false positives per microlitre. Furthermore, the system showed a sensitivity of 75% at the cell level, enabling the detection of low parasite densities in a fast and easy-to-use manner. A field trial in Chittagong (Bangladesh) indicated that future work should primarily focus on improving the filling process of the cartridge and the focus control part of the scanner.

Red cell quinine concentrations in falciparum malaria.

Red cell concentrations of quinine were measured in cerebral malaria and in uncomplicated falciparum malaria both in the acute stage and 1 month after recovery. Red cell quinine elimination half times were significantly shorter than the corresponding plasma half times in cerebral malaria and convalescent uncomplicated cases (P less than 0.001), but not in acute uncomplicated cases (P = 0.12). The ratio of red cell:plasma concentration fell progressively from the 2nd day (0.49) to the 6th day (0.26) of treatment. On admission this ratio was positively correlated with the blood parasite count (P = 0.02). Red cell quinine concentration may be relevant to the assessment of drug resistance, and to the design of treatment regimens.

Binding of quinine to plasma proteins in falciparum malaria.

Plasma protein binding of quinine was measured in 12 patients with cerebral malaria on the first and seventh day of treatment, and in 7 patients with uncomplicated falciparum malaria on admission and also one month later. Binding was significantly higher and therefore the proportion of free drug was lower in cerebral malaria patients (free: total quinine concentration; 7.2 +/- 3.5%, mean +/- SD, on admission; 7.4 +/- 5.3% on day 7) compared with uncomplicated malaria patients on admission (10.2 +/- 5.8%) or following recovery (11.0 +/- 5.5%, n = 6) P = 0.011. Binding was significantly correlated with the red cell/total concentration ratio r = 0.56, P less than 0.0001. The ratio of cerebrospinal fluid to free (unbound) plasma quinine was 0.55 +/- 0.33 which suggests that quinine does not freely cross the blood brain barrier. These findings are relevant to the interpretation of total plasma or serum concentration, and may explain the rarity of serious quinine toxicity in severe falciparum malaria.

Short report: Rosette formation in Plasmodium ovale infection.

Red blood cells infected by mature stages of Plasmodium ovale obtained from a 56-year-old Thai patient formed rosettes readily with uninfected erythrocytes. Ex vivo, the ring stage-infected erythrocytes matured well under the in vitro conditions used for P. falciparum culture, and the infected erythrocytes formed rosettes when the parasites became mature trophozoites. These rosettes were stable and remained intact until completion of schizogony. Plasmodium ovale rosettes were similar to those formed by P. falciparum- and P. vivax-infected erythrocytes. Rosette formation appears to be a common property of three species of human plasmodia.

Pharmacokinetics of three commercial antivenoms in patients envenomed by the Malayan pit viper, Calloselasma rhodostoma, in Thailand.

The pharmacokinetics of 3 monospecific antivenoms were compared in patients envenomed by the Malayan pit viper, Calloselasma rhodostoma. There was a biphasic decline in serum concentrations following intravenous administration. The initial rapid decline was attributable to the formation of venom-antivenom complexes, as the fall in antivenom during this phase was positively correlated with the initial venom concentration (P = 0.045). The total apparent volume of distribution for each antivenom was 1.5-3 times larger than that of the central compartment, which suggests some tissue distribution in addition to complex formation. This was marked for antivenom from the Government Pharmaceutical Organization of Thailand which contained mostly F(ab)2 fragments. The terminal elimination half time was shorter for Twyford antivenom of caprine origin. Systemic clearance was lower for Thai Red Cross antivenom. In 8 of the 26 patients who experienced recurrence of non-clotting blood after initial response to antivenom, serial measurements of plasma venom and antivenom concentrations revealed that recurrence of venom antigenemia and non-clotting blood bore no direct relation to the elimination half-life of the antivenom used, but non-clotting blood recurred when serum antivenom levels fell below 10-20% of the total given. There is no substitute for close monitoring of envenomed patients so that indications for further antivenom can be detected promptly.

Prognostic significance of reduced red blood cell deformability in severe falciparum malaria.

Severe falciparum malaria is associated with microvascular obstruction resulting from sequestration of erythrocytes containing mature stages of the parasite. Since reduced red blood cell deformability (RBC-D) can contribute to impaired microcirculatory flow, RBC-D was measured in 23 patients with severe falciparum malaria (seven of whom subsequently died), 30 patients with uncomplicated malaria, and 17 healthy controls. The RBC-D, measured by ektacytometry, was significantly reduced in severe malaria and was particularly low in all fatal cases. At a low shear stress of 1.7 Pascal (Pa), a red blood cell elongation index less than 0.21 on admission to the hospital predicted fatal outcome with a sensitivity of 100% (confidence interval [CI] = 59-100%) and a specificity of 88% (CI = 61-98%). The reduction in the RBC-D appeared to result mainly from changes in unparasitized erythrocytes. Reduced deformability of unparasitized red blood cells in severe malaria may contribute to impaired microcirculatory flow and a fatal outcome in severe falciparum malaria.

Red blood cell deformability as a predictor of anemia in severe falciparum malaria.

Decreased erythropoiesis and increased clearance of both parasitized and noninfected erythrocytes both contribute to the pathogenesis of anemia in falciparum malaria. Erythrocytes with reduced deformability are more likely to be cleared from the circulation by the spleen, a process that is augmented in acute malaria. Using a laser diffraction technique, we measured red blood cell (RBC) deformability over a range of shear stresses and related this to the severity of anemia in 36 adults with severe falciparum malaria. The RBC deformability at a high shear stress of 30 Pa, similar to that encountered in the splenic sinusoids, showed a significant positive correlation with the nadir in hemoglobin concentration during hospitalization (r = 0.49, P < 0.002). Exclusion of five patients with microcytic anemia strengthened this relationship (r = 0.64, P < 0.001). Reduction in RBC deformability resulted mainly from changes in unparasitized erythrocytes. Reduced deformability of uninfected erythrocytes at high shear stresses and subsequent splenic removal of these cells may be an important contributor to the anemia of severe malaria.

Nitric oxides in plasma, urine, and cerebrospinal fluid in patients with severe falciparum malaria.

It has been suggested that nitric oxide (NO) plays an important role in the pathogenesis of severe falciparum malaria. Since NO has a very short half-life, nitrate and nitrite (NOx) levels, stable metabolites of NO, are used as measures of NO production. We measured plasma NOx levels in 24 adults with severe falciparum malaria on the Thai-Burmese border. After correction for renal function, there was no correlation between plasma NOx levels, or the total amount of NOx excreted in the urine, and disease severity. Plasma NOx levels decreased after the first 48 hr in all patients (P = 0.007), suggesting decreased NO production. The NOx levels in cerebrospinal fluid (CSF) correlated well with plasma NOx levels, but these did not show a correlation with coma depth, and were not significantly different from those in a healthy control group. These findings do not support the hypothesis that excessive NO production contributes to the pathogenesis of severe falciparum malaria. However, local changes in NO production, e.g., in the central nervous system, might not be reflected in the total NOx production or NOx levels in the CSF.

Relation of the stage of parasite development in the peripheral blood to prognosis in severe falciparum malaria.

Admission blood films from 72 patients who died of severe falciparum malaria (50 Thai adults, 22 Gambian children) were matched retrospectively for parasitaemia with equal numbers of survivors. The peripheral blood parasites from fatal cases were more mature than those from survivors. Tiny rings (TR) comprised > 50% of parasites in 47/72 (65%) survivors but only 12/72 (17%) of fatal cases (P < 0.001). Parasites containing visible pigment (MTS: mature trophozoites and schizonts) comprised < 20% of the total parasite count in 10/72 (14%) survivors compared with 31/72 (43%) fatal cases (P < 0.001). Of the 39 patients with > 10(4) MTS/microL, 30 (81%) died. These findings were confirmed in a prospective study of 279 adult Thai patients admitted sequentially with acute falciparum malaria. Only 4 of the 19 fatal cases (21%) had > 50% TR, compared with 130 of 260 (50%) survivors, whereas > 20% MTS were found in 10/19 (53%) fatal cases, compared with 28/108 (27%) severe malaria survivors, and 26/155 (17%) patients with moderately severe malaria (P = 0.001). As a predictor of fatal outcome, the finding of either > 10(4) MTS/microL or > 5 x 10(5) parasites/microL in severe malaria had a sensitivity of 90% (95% confidence interval [CI] = 75-97%) and a specificity of 72% (95% CI = 59-86%). These observations are consistent with the hypothesis that a predominance of mature parasites in the peripheral blood reflects a greater sequestered biomass, and thus more severe disease. Simple microscopical assessment of parasite maturity on an admission blood slide provides important pathophysiological and prognostic information in severe falciparum malaria.

Quinine pharmacokinetics in cerebral malaria: predicted plasma concentrations after rapid intravenous loading using a two-compartment model.

To investigate the toxic potential of rapid intravenous quinine administration in severe malaria, the pharmacokinetic properties of low-dose quinine dihydrochloride injection (4 mg/kg body weight, equivalent to 3.3 mg base/kg) followed one hour later by infusion of 16 mg/kg over 3 h were studied in 7 patients with cerebral malaria. Plasma quinine concentrations closely followed a bi-exponential decline. Both the volumes of the central compartment (mean +/- SD: 0.17 +/- 0.10 litre/kg) and total volumes of distribution (0.74 +/- 0.30 litre/kg) were significantly smaller than those previously reported for healthy subjects. Based on the derived pharmacokinetic parameters, predicted plasma quinine concentrations following intravenous injection of the standard therapeutic dose (10 mg salt/kg) over 10-20 min are potentially toxic in severe malaria. Further reductions in administration time would produce disproportionately higher plasma quinine concentrations, especially as the distribution half-time (2.3 +/- 3.2 min) is approached. A theoretical regimen designed to achieve therapeutic, non-toxic plasma quinine concentrations promptly would be 7.0 mg quinine dihydrochloride/kg over 30 min. A subsequent maintenance infusion of 10 mg/kg over 4 h would allow for drug elimination and acute changes in pharmacokinetic parameters due to resuscitation and rehydration.

Ex-vivo short-term culture and developmental assessment of Plasmodium vivax.

A simple reproducible method for short-term ex-vivo Plasmodium vivax culture is presented in which glucose, ascorbic acid, thiamine, hypoxanthine, and 50% human AB+ serum are added to the standard P. falciparum in-vitro culture medium. Culture of freshly obtained blood samples from patients with acute vivax malaria with > 0.5% parasitaemia resulted in > 95% complete schizogony. Culture could be continued for 5-6 cycles without the addition of red cells. Criteria for staging the erythrocytic development of P. vivax in the first schizogonic cycle based on synchronous ex-vivo culture are presented. The asexual cycle was divided into 7 morphological stages: tiny ring (0-6 h), small ring (6-12 h), large ring (12-18 h), early trophozoite (18-28 h), late trophozoite (28-36 h), early schizont (36-42 h) and mature schizont (42-48 h). This simple method of culturing P. vivax ex vivo is suitable for antimalarial susceptibility and immunoparasitology studies.

A simple method for assessing quinine pre-treatment in acute malaria.

Administration of a loading dose of quinine in severe malaria may be dangerous if therapeutic blood concentrations are already present because of previous treatment. To assess the reliability of the history of pretreatment we conducted a prospective study of 379 adult patients with acute falciparum malaria admitted to a hospital in western Thailand. Admission plasma concentrations of quinine were measured by high performance liquid chromatography (HPLC), and compared with the patients' history of previous quinine treatment. The sensitivity of the history was 59% (95%) confidence interval [CI] 49-69%), the specificity was 79% (95% CI 74-84%), the positive predictive value was 53%, and the negative predictive value 82%. A rapid (10 min semi-quantitative estimate of plasma quinine concentrations, based on simple 'dipstick' method using a quinine-specific monoclonal antibody, proved considerably more sensitive and specific. The correlation between the dipstick estimate and the subsequent HPLC measurement of plasma quinine concentration was 0.85 (n = 404; P < 0.0001). In 404 admission samples, the negative predictive value of the dipstick estimate for plasma quinine concentrations > 1 microgram/mL was 100%. In Thailand the history of previous quinine treatment given by the patients or their relatives was unreliable but the quinine dipstick provided a simple and rapid means of assessment of quinine pre-treatment in acute falciparum malaria.

Red cell selectivity in malaria: a study of multiple-infected erythrocytes.

To characterize red cell susceptibility to invasion in malaria, a selectivity index (SI) was calculated as the ratio of observed number of multiple-infected red cells to that expected from a random process (Poisson distribution). In patients with falciparum malaria (n = 100) SI decreased with increasing parasitaemia (P < 0.001), and correlated inversely with plasma lactate concentrations, chosen prospectively as a measure of disease severity (r = -0.36, P < 0.001). For parasitaemias < 5%, the SI was lower in patients with severe malaria (geometric mean 1.35; 95% confidence interval 1.01-1.80) than in uncomplicated malaria (2.31; 1.89-2.81; P = 0.003), despite similar parasite counts. The geometric mean (range) SI in vivax malaria (n = 20), 7.69 (1.67, 29.75), was significantly greater than that in falciparum malaria at comparable parasitaemias (< or = 2%), 2.44 (0.45, 14.05), P < 0.001, suggesting that about 13% of circulating erythrocytes were susceptible to invasion by Plasmodium vivax. This translates into susceptibility for about 2 weeks after emergence from the bone marrow, if age is the sole determinant of this process. In falciparum malaria selectivity was inversely proportional to severity; lack of selectivity could reflect either a 'favourable' host red cell phenotype, or an indiscriminate parasite population. Both are dangerous for the host.

Arboreal green pit vipers (genus Trimeresurus) of South-East Asia: bites by T. albolabris and T. macrops in Thailand and a review of the literature.

In Thailand 29 patients were proved to have been bitten by arboreal green pit vipers: 24 by Trimeresurus albolabris and 5 by T. macrops. They were studied in order to define the clinical effects of envenoming, to characterize the haemostatic abnormalities and assess the efficacy of Thai Red Cross antivenom. T. macrops caused only local painful swelling, neutrophil leucocytosis and thrombocytopenia. T. albolabris caused more severe envenoming with local blistering and necrosis, shock, spontaneous systemic bleeding, defibrination, thrombocytopenia and leucocytosis. There was no evidence of disseminated intravascular coagulation, but fibrinolytic activity was increased. Platelet function was normal. The product of admission venom antigen concentration and the delay between bite and admission was significantly higher in defibrinated patients than in those without severe coagulopathy. Antivenom (5 ampoules intravenously) restored blood coagulability, but there was persistent venom antigenaemia, associated in some cases with recurrent coagulopathy. The literature on bites by south Asian green pit vipers of the genus Trimeresurus is reviewed; these bites are common medical problems and causes of morbidity. The identification of individual species is difficult, but may be important if antivenom is to be improved and used appropriately.

A comparison of artesunate alone with combined artesunate and quinine in the parenteral treatment of acute falciparum malaria.

In some areas clinicians have combined parenteral artesunate and quinine in the belief that the 2 drugs would be additive or synergistic in severe malaria. A randomized comparison of the effectiveness of intravenous (i.v.) artesunate versus i.v. artesunate and i.v. quinine together on parasite clearance was conducted in 1998/99 amongst 69 patients with uncomplicated and severe Plasmodium falciparum malaria in western Thailand. The parasite clearance time did not differ significantly between the 2 treatment groups (P = 0.12), but adverse events were significantly more frequent in the artesunate plus quinine group (P = 0.05). Quinine did not have a significant antipyretic effect and artesunate did not affect the electrocardiographic QTc interval. There is no benefit evident from combining parenteral administration of these 2 antimalarial drugs in the acute phase of treatment.

Semi-quantitative measurement of Plasmodium falciparum antigen PfHRP2 in blood and plasma.

Plasmodium falciparum histidine rich protein 2 (PfHRP2) antigen was measured semi-quantitatively in whole blood, plasma, and supernatants and red blood cells of cultures in vitro using the dipstick ParaSight-F test and also by a quantitative antigen-capture enzyme-linked immunosorbent assay (ELISA). In vitro, PfHRP2 was secreted mainly during the second half of the asexual cycle with a marked rise during schizont development and rupture. The total PfHRP2 secreted before schizogony corresponded to approximately 4% of that contained in the red blood cells. In samples from 55 patients with acute falciparum malaria, the level of detection by ELISA corresponded to parasitaemias of 100/microL for whole blood and 1600/microL for separated plasma. Whole blood PfHRP2 levels were correlated significantly with admission parasitaemia (r = 0.76, P < 0.0001) and the stage of parasite development (r = 0.43, P < 0.01). Although whole blood PfHRP2 concentrations were higher in severe malaria, plasma concentrations of PfHRP2 were considerably higher in severe malaria (median titre 1:320, range zero to 1:1280) than in uncomplicated malaria (median titre 1:5, range zero to 1:80; P < 0.0001). The ratio of whole blood to plasma PfHRP2 was lower in severe than in uncomplicated malaria (median 4, range 0.25 to 256, versus 64, range 4 to 1280; P < 0.0001). With plasma samples the intensity of colour change on the dipstick correlated well with more precise measurement of optical density in the ELISA (r = 0.88, P < 0.0001). These results suggest that measurement of PfHRP2 in plasma could provide an alternative approach to the assessment of the parasite biomass, and thus prognosis, in severe malaria, and that this could be done simply by using the currently available dipsticks.

Evidence for continued two-brood replication of Plasmodium falciparum in vivo during quinine treatment.

To investigate the relationship between fever and parasite clearance in falciparum malaria, we studied 54 adults with Plasmodium falciparum infections who were all treated with quinine. The median oral temperature profile showed peaks at 24 h intervals during the first 3 days. Although there was no equivalent pattern evident in the median parasite clearance curve, we hypothesize that small numbers of two distinct parasite broods continued to develop in antiphase through schizogony despite quinine therapy. These data are consistent with previous reports of two dominant broods in untreated humans and monkeys infected with P. falciparum, and highlight the need for an adequate duration of quinine treatment.

Erythrocyte survival in severe falciparum malaria.

Erythrocyte survival was studied in 17 Thai patients (10 males, 7 females; aged 13-57 years) with severe falciparum malaria. To ensure radioisotopic labelling of cells before bone marrow recovery and survival analysis under near-steady state conditions, 51Cr labelling of autologous erythrocytes was performed at the time of admission (0 h) and calculation of mean cell lifespan (MCL) was based on semilogarithmic plots of corrected counts from 60 h onwards. Five patients received blood transfusions, all within 48 h of admission. The overall mean (+/- S.D.) MCL was short (44.1 +/- 21.7 days). Nontransfused patients had similar MCL values (43.6 +/- 20.4) to those of transfused patients (45.5 +/- 27.3 days, p greater than 0.8). Patients with and without palpable splenomegaly had MCL values which were not significantly different (54.1 +/- 28.8 vs. 37.2 +/- 12.3 days respectively, p greater than 0.1). There was no association between admission haematocrit or peripheral parasitaemia and MCL (p greater than 0.2 in each case), but there was an inverse correlation between total serum bilirubin and MCL (r = -0.49, p less than 0.025). There is accelerated destruction of non-parasitised erythrocytes in severe malaria resulting in a mean MCL that is half that found previously in healthy Thai volunteers (89.6 +/- 13.1 days, p less than 0.001) and significantly shorter than that reported previously in Thai patients with uncomplicated P. falciparum infections studied after parasite clearance (56.8 +/- 10.2 days, p less than 0.05).

Plasmodium malariae in Bangladesh.

We describe a 32-year-old Bangladeshi male presenting with severe malaria caused by a mono-infection with Plasmodium malariae. Rosetting of infected and uninfected erythrocytes, a putative virulence factor in falciparum malaria, was observed in the blood slide. Severe disease caused by P. malariae is extremely rare. The patient made a rapid recovery with intravenous quinine treatment.