Development and validation of multiplex real-time PCR for simultaneous detection of six bacterial pathogens causing lower respiratory tract infections and antimicrobial resistance genes.

BACKGROUND: Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae and Staphylococcus aureus are major bacterial causes of lower respiratory tract infections (LRTIs) globally, leading to substantial morbidity and mortality. The rapid increase of antimicrobial resistance (AMR) in these pathogens poses significant challenges for their effective antibiotic therapy. In low-resourced settings, patients with LRTIs are prescribed antibiotics empirically while awaiting several days for culture results. Rapid pathogen and AMR gene detection could prompt optimal antibiotic use and improve outcomes. METHODS: Here, we developed multiplex quantitative real-time PCR using EvaGreen dye and melting curve analysis to rapidly identify six major pathogens and fourteen AMR genes directly from respiratory samples. The reproducibility, linearity, limit of detection (LOD) of real-time PCR assays for pathogen detection were evaluated using DNA control mixes and spiked tracheal aspirate. The performance of RT-PCR assays was subsequently compared with the gold standard, conventional culture on 50 tracheal aspirate and sputum specimens of ICU patients. RESULTS: The sensitivity of RT-PCR assays was 100% for K. pneumoniae, A. baumannii, P. aeruginosa, E. coli and 63.6% for S. aureus and the specificity ranged from 87.5% to 97.6%. The kappa correlation values of all pathogens between the two methods varied from 0.63 to 0.95. The limit of detection of target bacteria was 1600 CFU/ml. The quantitative results from the PCR assays demonstrated 100% concordance with quantitative culture of tracheal aspirates. Compared to culture, PCR assays exhibited higher sensitivity in detecting mixed infections and S. pneumoniae. There was a high level of concordance between the detection of AMR gene and AMR phenotype in single infections. CONCLUSIONS: Our multiplex quantitative RT-PCR assays are fast and simple, but sensitive and specific in detecting six bacterial pathogens of LRTIs and their antimicrobial resistance genes and should be further evaluated for clinical utility.

The influence of smoking on oral neutrophils and matrix metalloproteinase-8 in periodontitis patients before and after nonsurgical treatment.

Objective: To evaluate and compare the oral neutrophil numbers (ONN) in saliva, the level of matrix metalloproteinase-8 (MMP-8) in gingival crevicular fluid (GCF) and the periodontal parameters in smokers versus non-smokers with periodontitis, before and after nonsurgical periodontal treatment (NSPT). Materials and method: 40 chronic periodontitis patients including 20 smokers and 20 non-smokers were enrolled in this quasi-experimental study. All patients were received the NSPT included instructing oral hygiene, scaling and root planing. At baseline (T0) and after NSPT 1 month (T1) and 3 months (T3), all patients were assessed for salivary ONN, GCF MMP-8, and clinical parameters like plaque index (PlI), gingival index (GI), bleeding on probing (BOP), probing pocket depth (PPD) and clinical attachment loss (CAL). The differences between the two groups were analyzed using the independent sample t-test and the Mann-Whitney U test; and the differences between T0, T1 and T3 of each group were analyzed with paired-samples t-test and Wilcoxon signed-rank test. The level of significance was set at 0.05. Results: The ONN was significantly less in smokers than in non-smokers although there was no significant difference in other parameters between the two groups at baseline (p > 0.05). All clinical periodontal parameters reduced significantly after 1 month and 3 months of NSPT in both groups (p < 0.01). PPD of non-smokers was significantly lower than those of smokers at T1 and T3. ONN and MMP-8 level showed a significant decrease in non-smoking subjects, while there was no significant difference in smoking ones after NSPT (T1 and T3). At 1 month after treatment, ONN tended to reduce in non-smokers whereas to increase in smokers significantly. Conclusion: Smoking reduced ONN, impaired treatment effect in reducing PPD, and changed the MMP-8 level in gingival crevicular fluid to NSPT. Trial registration: Identifier NCT04974502 in CLinicalTrials.gov.

Plastic waste as a valuable resource: strategy to remove heavy metals from wastewater in bench scale application.

Single-use plastic waste is gradually considered a potential material for circular economy. Ion exchange resin obtained from polystyrene waste by sulfonating with H2SO4 was used for heavy metal removal from electroplating wastewater. Batch mode experiments of Cu2+, Zn2+, and Cd2+ were carried out to determine effect of pH, initial concentration, equilibrium time, and the isotherm and kinetic parameters; the stability of the resin in continuous operation was then evaluated. Finally, the longevity of the resin after being exhausted was explored. The results indicated that at pH 6, a pseudo-second-order kinetic model was applicable to describe adsorption of studied heavy metals by sulfonated polystyrene with adsorption capacities of 7.48 mg Cu2+/g, 7.23 mg Zn2+/g, and 6.50 mg Cd2+/g, respectively. Moreover, the ion exchange process between sulfonated polystyrene resin and Cu2+, Zn2+, Cd2+ ions followed the Langmuir isotherm adsorption model with R2 higher than 96%. The continuous fixed-bed column in conditions of a sulfonated polystyrene mass of 500 g, and a flow rate of 2.2 L/h was investigated for an influent solution with known initial concentration of 20 mg/L. Thomas and Yoon-Nelson models were tested with regression analysis. When being exhausted, the sulfonated polystyrene was regenerated by NaCl in 10 min with ratio 5 mL of NaCl 2 M per 1 g saturated resins. After 4 times regeneration, the heavy metal removal efficiency of sulfonated polystyrene was reduced to 50%. These aforementioned results can figure out that by sulfonating polystyrene waste to synthesize ion exchanging materials, this method is technically efficient and environmentally friendly to achieve sustainability.

Accumulation of copper and cadmium in soil-rice systems in terrace and lowland paddies of the Red River basin, Vietnam: the possible regulatory role of silicon.

Rice production in floodplain deltas is currently vulnerable to climate change and contamination from anthropogenic activities. The relocation of rice production to upland regions could be an option for increasing the sustainability of rice production. Our study evaluated the spatial patterning of heavy metals, i.e., copper (Cu) and cadmium (Cd), in rice along a topogradient from terrace to lowland areas in the Red River basin. The dataset obtained from the analysis of 61 farm sites throughout the whole basin indicated a large discrepancy in the Cu and Cd contents in rice grains from terrace and lowland paddies. While Cu and Cd were not found in most of the rice grain samples from the terrace paddies, the median Cu and Cd contents of the lowland paddy rice were 1.895 and 0.033 mg kg-1, respectively. Assessing the relationship of Cu and Cd in the soil-rice system to soil properties revealed possible correlations between soil available silicon (Si) and the Cu and Cd contents in rice grain. The enrichment of Si in rice plants likely reduces the translocation of Cu and Cd from soil to grain. Therefore, management of the Si supply, particularly in lowland paddies, should be highlighted as a way to reduce dietary intake of Cu and Cd.

Accumulation and potential health risks of cadmium, lead and arsenic in vegetables grown near mining sites in Northern Vietnam.

The effect of environmental pollution on the safety of vegetable crops is a serious global public health issue. This study was conducted to assess heavy metal concentrations in soil, irrigation water, and 21 local vegetable species collected from four sites near mining activities and one control site in Northern Vietnam. Soils from vegetable fields in the mining areas were contaminated with cadmium (Cd), lead (Pb), and arsenic (As), while irrigation water was contaminated with Pb. Average concentrations of Pb and As in fresh vegetable samples collected at the four mining sites exceeded maximum levels (MLs) set by international food standards for Pb (70.6 % of vegetable samples) and As (44.1 % of vegetable samples), while average Cd concentrations in vegetables at all sites were below the MLs of 0.2. The average total target hazard quotient (TTHQ) across all vegetable species sampled was higher than the safety threshold of 1.0, indicating a health risk. Based on the weight of evidence, we find that cultivation of vegetables in the studied mining sites is an important risk contributor for local residents' health.

Pellet reactor pretreatment: a feasible method to reduce scaling in bipolar membrane electrodialysis.

This study aims to evaluate the feasibility of a pellet reactor in reducing the scaling potential in electrodialysis with bipolar membranes for water containing a high concentration of calcium by adding sodium carbonate to precipitate carbonate as calcium carbonate on granular seed material. The optimized operating condition obtained at pH 11.1, and a ratio of [CO3(2-)]:[Ca(2+)]=1.2:1 enabled to obtain 90% efficiency of calcium removal from real water. The efficiency of scaling potential removal was validated by comparing the scaling level on the membrane surface of two electrodialysis batches of a washing water, with and without pretreatment. For the latter, scalants were found at both sides of the cation exchange membrane (FKB), diluate and base sides, identified as calcium and magnesium precipitates. Furthermore, there was also a severe scaling effect at the base side of the bipolar membrane (FBM). However, a different observation was found for the pretreated water. SEM and elemental analysis for both FKB and FBM membranes demonstrated less scaling on both membrane surfaces.

Calibration of a passive sampling device for time-integrated sampling of hydrophilic herbicides in aquatic environments.

Two types of solid-phase materials, a styrenedivinylbenzene copolymer sorbent (embedded in a SDB-XC Empore disk) and a styrenedivinylbenzene copolymer sorbent modified with sulfonic acid functional groups (embedded in a SDB-RPS Empore disk), were compared as a receiving phase in a passive sampling device for monitoring polar pesticides. The SDB-XC Empore disk was selected for further evaluation, overlayed with either a polysulfone or a polyethersulfone diffusion membrane. The target herbicides included five nonionized herbicides (simazine, atrazine, diuron, clomazone, and metolachlor) and four phenoxy acid herbicides (dicamba, (2,4-dichlorophenoxy)acetic acid [2,4-D], (4-chloro-2-methylphenoxy)acetic acid [MCPA], and triclopyr) with log octanol/water partition coefficient (log K(OW)) values of less than three in water. Uptake of these herbicides generally was higher into a device constructed of a SDB-XC Empore disk as a receiving phase covered with a polyethersulfone membrane compared to a similar device covered with a polysulfone membrane. Using the device with a SDB-XC Empore disk covered with a polyethersulfone membrane, linear uptake of simazine, atrazine, diuron, clomazone, and metolachlor was observed for up to 21 d, and daily sampling rates of the herbicides from water in a laboratory flow-through system were determined. The uptake rate of each nonionized herbicide by the Empore disk-based passive sampler was linearly proportional to its concentration in the water, and the sampling rate was independent of the water concentrations over the 21-d period. Uptake of the phenoxy acid herbicides (2,4-D, MCPA, and triclopyr) obeyed first-order kinetics and rapidly reached equilibrium in the passive sampler after approximately 12 d of exposure. The Empore disk-based passive sampler displayed isotropic kinetics, with a release half-life for triclopyr of approximately 6 d.

Determination of commonly used polar herbicides in agricultural drainage waters in Australia by HPLC.

The present study describes the application of different extraction techniques for the preconcentration of ten commonly found acidic and non-acidic polar herbicides (2,4-D, atrazine, bensulfuron-methyl, clomazone, dicamba, diuron, MCPA, metolachlor, simazine and triclopyr) in the aqueous environment. Liquid-liquid extraction (LLE) with dichloromethane, solid-phase extraction (SPE) using Oasis HLB cartridges or SBD-XC Empore disks were compared for extraction efficiency of these herbicides in different matrices, especially water samples from contaminated agricultural drainage water containing high concentrations of particulate matter. Herbicides were separated and quantified by high performance liquid chromatography (HPLC) with an ultraviolet detector. SPE using SDB-XC Empore disks was applied to determine target herbicides in the Murrumbidgee Irrigation Area (NSW, Australia) during a two-week survey from October 2005 to November 2005. The daily aqueous concentrations of herbicides from 24-h composite samples detected at two sites increased after run-off from a storm event and were in the range of: 0.1-17.8 microg l(-1), < 0.1-0.9 microg l(-1) and 0.2-17.8 microg l(-1) at site 1; < 0.1-3.5 microg l(-1), < 0.1-0.2 microg l(-1) and < 0.2-3.2 microg l(-1) at site 2 for simazine, atrazine and diuron, respectively.

Optimisation of the separation of herbicides by linear gradient high performance liquid chromatography utilising artificial neural networks.

An artificial neural network (ANN) was employed to model the chromatographic response surface for the linear gradient separation of 10 herbicides that are commonly detected in storm run-off water in agricultural catchments. The herbicides (dicamba, simazine, 2,4-D, MCPA, triclopyr, atrazine, diuron, clomazone, bensulfuron-methyl and metolachlor) were separated using reverse phase high performance liquid chromatography and detected with a photodiode array detector. The ANN was trained using the pH of the mobile phase and the slope of the acetonitrile/water gradient as input variables. A total of nine experiments were required to generate sufficient data to train the ANN to accurately describe the retention times of each of the herbicides within a defined experimental space of mobile phase pH range 3.0-4.8 and linear gradient slope 1-4% acetonitrile/min. The modelled chromatographic response surface was then used to determine the optimum separation within the experimental space. This approach allowed the rapid determination of experimental conditions for baseline resolution of all 10 herbicides. Illustrative examples of determination of these components in Milli-Q water, Sydney mains water and natural water samples spiked at 0.5-1mug/L are shown. Recoveries were over 70% for solid-phase extraction using Waters Oasis((R)) HLB 6cm(3) cartridges.

Combinations of artemisinin and quinine for uncomplicated falciparum malaria: efficacy and pharmacodynamics.

Combinations of artemisinin and quinine for uncomplicated falciparum malaria were studied. A total of 268 patients were randomized to 7 days of quinine at 10 mg/kg of body weight three times a day (Q) or to artemisinin at 20 mg/kg of body weight followed by 3 (AQ3) or 5 (AQ5) days of quinine. Recrudescence rates were 16, 38, and 15% for the Q, AQ3, and AQ5 groups, respectively (P < 0.001). Recrudescence was associated with shorter parasite clearance time (PCT) and longer treatment after the blood smear had become negative (eradication time). However, classification of patients to outcome-recrudescence or radical cure-was correct in only 77% of patients. The population kinetics of the parasitemia was estimated with nonlinear mixed-effect models. Several models were tested, but the best model was a monoexponential decline of the parasitemia in which the mean parasite elimination half-life was shorter after artemisinin (5.1 h; 95% confidence interval [CI], 4.9 to 5.2 h) than after quinine (8.0 h [95% CI, 7.5 to 8.3 h]). Attempts to simulate the initial increase of the parasitemia did not result in better models with a biologically plausible interpretation. Recrudescence was associated with slower parasite clearance and a higher simulated terminal parasitemia (P(term)). The classification of patients to outcome groups based on P(term) was correct in 78% of patients. The data suggest that parasite strains with reduced sensitivity to quinine are prevalent in Vietnam, with slower parasite clearance and consequent recrudescence. A single dose of artemisinin induces rapid parasite reduction and lowers the value of P(term), but to prevent recrudescence, this should be followed by quinine for at least 3 days after parasite clearance, or 5 days in total.

Glucose metabolism in severe malaria: minimal model analysis of the intravenous glucose tolerance test incorporating a stable glucose label.

Basal plasma glucose is usually increased in uncomplicated malaria, implying insulin resistance. If the infection progresses, the risk of hypoglycemia will increase as host glucose production becomes insufficient for host/parasite demand. To assess the relative contribution of insulin-mediated and non-insulin-mediated glucose disposal to plasma glucose levels in severe malaria, we studied six healthy controls (two males and four females; mean age, 38 years) and eight patients with complicated falciparum malaria (five males and three females; mean age, 31 years) who had a frequently sampled intravenous glucose tolerance test (FSIVGTT) in which 10% of the dextrose bolus was 6,6-D2-glucose. The minimal model was applied to native and labeled plasma glucose and serum insulin profiles over 4 hours postinjection. Basal plasma glucose concentrations in the patients were significantly greater than in the controls (median [range], 6.1 [2.1 to 8.5] v 4.3 [3.9 to 4.7] mmol/L, P = .03). Malaria-associated insulin resistance was confirmed by a lower insulin sensitivity index (SI) in patients (5.6 [2.4 to 17.4] v 16.0 [2.5 to 22.3] x 10(-4).min-1 per microU/mL in controls, P = .026). Glucose effectiveness ([SG] the ability of glucose to reduce its own plasma concentration) was higher in the patients (0.015 [0.006 to 0.024] v 0.008 [0.007 to 0.010] min-1 in controls, p = .019). Glucose disappearance at basal concentration was increased by a median of 42% in severe malaria patients, with the insulin-independent component comprising 81%, versus 67% in controls. Indices of beta-cell function were normal in malaria patients. These data demonstrate that basal plasma glucose utilization is increased approximately 50% in severe malaria, consistent with previously published isotope-turnover studies. Altered SG plays a major role. Prevention and treatment of early hypoglycemia should be based on adequate glucose replacement. Strategies that reduce insulin secretion or effects appear to be of minor importance.

Efficacy and tolerance of artemisinin in short combination regimens for the treatment of uncomplicated falciparum malaria.

Two oral regimens comprising a single dose of 20 mg/kg of artemisinin followed by three days of quinine, 10 mg/kg three times a day (AQ), or doxycycline, 4 mg/kg once a day (AD), were compared with a standard seven-day course of oral quinine, 10 mg/kg three times a day (Q), in the treatment of uncomplicated falciparum malaria. Of 161 treated patients, 157 could be included in the analysis. The mean +/- SD parasite clearance time was 43 +/- 14 hr for AQ and 41 +/- 19 hr for AD, and significantly longer for quinine: 66 +/- 24 hr (P = 0.0001). Treatment failure occurred in one Q and in 3 AD patients. The recrudescence rate was 16% for Q, 28% for AQ, and significantly worse for AD: 67% (P = 0.0001). Adverse effects were mainly limited to cinchonism. The conclusion is that a seven-day course of quinine is still effective in the initial treatment of uncomplicated falciparum malaria in Vietnam, but one should pay attention to possible recrudescence. The addition of a single 20 mg/kg per os dose of artemisinin allows for shortening the duration of treatment, with faster parasite clearance, comparable efficacy, and better tolerance, but with no reduction of recrudescence. The combination of artemisinin with three days of doxycycline is also not effective in preventing recrudescence.

Selective high-performance liquid chromatographic determination of artesunate and alpha- and beta-dihydroartemisinin in patients with falciparum malaria.

A novel solid-phase extraction and a robust high-performance liquid chromatographic (HPLC) separation procedure for artesunate and alpha- and beta-dihydroartemisinin, using post-column alkali decomposition and UV detection is described. Extraction was performed with Bond-Elut Phenyl solid-phase extraction cartridges and analysis by HPLC was carried out using a Waters Symmetry C8 5-microns 150 x 3.9 mm I.D. column. The mobile phase was 50% acetonitrile in 0.1 M acetate buffer (pH 4.8) delivered at a flow-rate of 0.7 ml/min. The column eluate was mixed with 1.2 M potassium hydroxide in 90% methanol delivered at 0.3 ml/min, in a 1-ml reaction coil at 69 degrees C, to form UV-absorbing chromophores which were detected at 290 nm. The recovery of all analytes was greater than 80%. There was no significant difference in the peak-area ratio of alpha- and beta-dihydroartemisinin in plasma. Preliminary pharmacokinetic data from six adult Vietnamese patients who received 120 mg of artesunate by intravenous injection for the treatment of acute falciparum malaria are presented. Despite limited data, the mean half-life of artesunate was approximately 3.5 min while that for dihydroartemisinin was 34 min. These data confirm the relatively rapid clearance of both artesunate and its principle active metabolite, dihydroartemisinin.

The metabolism of platelet-activating factor in severe and cerebral malaria.

In order to examine the effects of platelet-activating factor (PAF) in complicated Plasmodium falciparum infections, plasma concentrations of lyso-PAF, stable metabolite and principal precursor of PAF, were measured in 25 Vietnamese adults with severe malaria. The concentration of PAF in the cerebrospinal fluid (CSF) was determined in a sub-group of 23 comatose patients and, together with that of lyso-PAF, in the plasma of 20 patients on recovery of consciousness. The concentration of lyso-PAF in the plasma was depressed on admission to hospital (median [range]; 21 [8-143] vs. 293 [215-410] ng/ml in 10 controls; P < 0.001). There was, however, no change in plasma activity of acetylhydrolase which converts PAF to lyso-PAF (P > 0.01 vs. controls) while simultaneous reduction in the concentration of lipoproteins associated with lyso-PAF were less than those of lyso-PAF per se in the plasma. The plasma concentration of lyso-PAF on admission was associated with parasitaemia and the concentration of serum triglycerides (rs = -0.42, P = 0.04 in each case), the latter being consistent with hepatic effects of PAF reported in previous studies. CSF concentrations of PAF on admission were low (2.3 [0.5-7.7] vs. 0.9 [0-2.5] ng/ml after recovery, P < 0.01) compared with values reported previously in bacterial meningitis. Plasma concentrations of lyso-PAF after recovery lay between admission and control values. While increased availability of PAF may reflect parasite burden and may modulate liver-mediated metabolic disturbances such as hypoglycaemia and lactic acidosis, the role of PAF in cerebral malaria is uncertain.

Mineral homoeostasis in acute renal failure complicating severe falciparum malaria.

Disturbances in calcium metabolism in acute renal failure (ARF) remain incompletely understood. Most data are from patients with rhabdomyolysis. As renal impairment commonly accompanies severe malaria in the absence of rhabdomyolysis, falciparum malaria provides an alternative model of mineral homoeostasis in ARF. We studied 25 Vietnamese subjects, aged 18-63 yr, with severe malaria and 10 controls. Fourteen patients had a serum creatinine level of 250 mumol/L or less during treatment (group 1), five developed ARF but were not dialyzed (group 2a), and six required dialysis (group 2b). Group 1 patients presented with mild hypocalcemia (mean +/- SD serum ionized calcium, 1.18 +/- 0.05 vs. 1.23 +/- 0.02 mmol/L in controls; P = 0.01) that persisted until discharge in the presence of normal serum phosphate, PTH, and vitamin D metabolite levels. Group 2 patients were more hypocalcemic on admission (1.10 +/- 0.08 mmol/L; P < 0.0001 vs. controls), especially those in group 2b whose serum ionized calcium fell to 0.88 +/- 0.13 mmol/L when renal dysfunction was maximal. In group 2 patients, the admission serum PTH level was raised (5.4 +/- 3.8 vs. 2.7 +/- 0.9 pmol/L in controls; P < 0.02) and changed reciprocally with calcemia. Significant rises in serum phosphate occurred only in group 2b patients who had depressed serum free 1,25-dihydroxyvitamin D levels throughout. Hypercalcemia did not accompany the diuretic phase of ARF. These data suggest that parathyroid gland dysfunction is a cause of hypocalcemia in severe malaria without ARF, as seen in group 1 patients; in patients with ARF, the effect of the combination of phosphate retention and altered vitamin D metabolism on skeletal PTH sensitivity is of prime significance.

Serum vitamin A and E concentrations in acute falciparum malaria: modulators or markers of severity?

1. To assess the association between vitamin A, vitamin E and the clinical course of severe malaria, serial morning blood samples were taken from 24 Vietnamese patients, aged 18-62 years, receiving intensive treatment for complicated Plasmodium falciparum infections. A single fasting blood sample was also taken from 10 control subjects aged 22-45 years. Serum retinol, carotene and vitamin E concentrations were measured by h.p.l.c. 2. Admission serum retinol concentration was depressed relative to that of the control subjects (0.69 +/- 0.35 versus 1.86 +/- 0.41 mumol/l mean +/- SD, P < 0.001) and correlated inversely with indices of hepatic function, but positively with the simultaneous serum creatinine concentration (P < 0.05). During the first week of treatment, serum retinol concentration increased in parallel with improving liver function, whereas serum creatinine concentration remained elevated in the majority of patients. Serum alpha- and beta-carotene concentrations remained depressed throughout. 3. Serum vitamin E concentration, corrected for total serum cholesterol concentration in the form of a ratio, was also depressed at presentation (3.1 +/- 1.8 x 10(3) versus 4.2 +/- 0.8 x 10(3) in control subjects; P < 0.05), but tended to be higher than the control value at the time of discharge (0.1 > P > 0.05); there was a significant correlation between admission ratio and parasite clearance time (P = 0.04). 4. On the basis of this and previous studies, vitamin A replacement could be considered in selected severely ill patients without renal impairment. As found previously in animal models, depressed vitamin E levels may have a beneficial effect on the course of malarial infection.

Differential sequestration of parasitized erythrocytes in the cerebrum and cerebellum in human cerebral malaria.

Sequestration of parasitized red blood cells (PRBC) in the microvessels results in impairment of microcirculation with organ dysfunction in complicated human Plasmodium falciparum malaria. In cerebral malaria patients, the percentage of small blood vessels with PRBC sequestration is higher in the brain than in other organs. The clinical severity of cerebral malaria depends on the level of PRBC sequestration in the brain. In our study, postmortem samples from cerebrum and cerebellum of 16 patients who died of P. falciparum malaria were examined and compared using light microscopy. In the cerebellum, the percentage of microvessels with PRBC sequestration was higher than that in the cerebrum. The difference in sequestration rates between cerebrum and cerebellum is statistically significant (P < 0.05). There is a higher degree of vascularity in the cerebellum (7 vessels/mm2) than in the cerebrum (5 vessels/mm2), and the difference is also statistically significant (P < 0.025). Perivascular hemorrhages also occur more frequently in the cerebellum than in the cerebrum. The results of this study, which show that differential sequestration of PRBC occurs in the microvessels of the cerebrum and cerebellum, explain the varied neurologic manifestations that result from cerebral and cerebellar dysfunction in human cerebral malaria. This study also reveals the necessity of postmortem histologic examination of the cerebellum in every suspected case of cerebral malaria.