In vitro susceptibility studies of Malaysian Plasmodium falciparum isolates and clones against schizontocidal drugs

Spasmodic dysphonia (SD) is at present defined as focal dystonia. Botulinum toxin (BT) injection is the treatment of choice for SD. BT is usually injected by a percutaneous route, but a direct, visually guided transoral approach has also been successful. It is not known whether percutaneous injection is as effective as the transoral approach. This article reviews our experience with both techniques of injection on 29 patients with adductor type SD. Since 1992, we have carried out 48 treatment sessions with the transoral technique and 76 treatment sessions with the percutaneous technique. Two patients did not respond to the percutaneous technique despite several attempts, but they did respond to the transoral approach. Globally, transoral technique was superior to percutaneous technique in terms of effectiveness (48 of 48 responses with transoral technique versus 61 of 76 responses with percutaneous approach, p < 0.01). Dosage of BT, duration, and side effects were similar with both techniques. This article also describes a simple, inexpensive device, composed of materials on hand at every hospital, that facilitates the transoral approach.     

Subacute Plasmodium falciparum malaria in 43 patients returning from areas with chloroquine in Africa

PURPOSE: To identify clinical and biological features of subacute falciparum malaria, risk factors, and to evaluate the efficacy of curative treatment. PATIENTS AND METHODS: Diagnostic criteria were the association of apyrexia, anemia, little or no parasitemia and a high titer of anti-Plasmodium antibodies. Forty-three cases were observed in subjects returning from chloroquine-resistant areas in Africa. They were matched with controls for age, country of residence and duration of stay. Controls were missionaries who attended our unit for a routine medical check-up during the study period. RESULTS: The clinical presentation and biological features were similar to "malarial cachexia", a condition mainly described in non-immune children in endemic areas. Splenomegaly was present in 58% of the patients. Biological features included little or no parasitemia, an overall decrease in the blood cell count, an increased erythrocyte sedimentation rate and a high titer of anti-Plasmodium antibodies. This syndrome was not correlated with the frequency of chloroquine resistance, the area of stay (urban or rural) or to the kind of chemoprophylaxis. CONCLUSIONS: This study describes subacute resistant falciparum malaria in patients who had prolonged stay in chloroquine-resistant areas of Africa associating splenomegaly, cytopenia and a low or absent parasitemia. Subacute chloroquine-resistant malaria could be due to host factors which remained to be determined by prospective immunological studies. Curative treatment with mefloquine is effective.     

Minimal variation in the Pfs28 ookinete antigen from Philippine field isolates of Plasmodium falciparum

The study examined therapists' accuracy in predicting the length of individual outpatient psychotherapy for 109 clients and attempted to identify variables associated with predicted and actual treatment lengths. The mean predicted treatment length (9.7 months) was significantly longer than the mean length of actual treatment (6.6 months). Therapists correctly predicted treatment length to the nearest month in 26 percent of the cases. Predictions were more accurate for older clients. Treatment tended to be shorter for clients with less than a high school education. Therapists more often predicted shorter treatments for clients with an adjustment disorder and those with less education. Predicting treatment length appears to be difficult.     

Allelic diversity at the merozoite surface protein-1 locus of Plasmodium falciparum in clinical isolates from the southwestern Brazilian Amazon

A rapid dynamic imaging technique based on polar k-space sampling is presented. A gain in temporal resolution is achieved by angular undersampling. A detailed analysis of the point spread function of angular undersampled polar imaging reveals a reduced diameter of the corresponding circular field of view. Under the assumption that dynamic changes are restricted to a local circular field of view, angular undersampled dynamic imaging allows the recording of rapid changes at high temporal and spatial resolution. The theoretical and experimental details of the technique are presented.     

Considering disparities in resistance of Plasmodium falciparum in Africa in chemoprevention decisions

OBJECTIVES: Assess the efficacy of preventive and curative treatments of imported malaria. METHODS: The in vitro drug susceptibility of mefloquine, chloroquine and cycloguanil was determined against African isolates of Plasmodium falciparum from imported malaria cases by an isotopic in vitro test or a genomic approach. RESULTS: Plasmodium falciparum resistance to mefloquine, chloroquine or to the dihydrofolate reductase inhibitor was present in 5.2%, 46% and 42% of isolates respectively. Plasmodium falciparum drug resistance to chloroquine or antifolinics was more frequent in permanent than in seasonal malarial transmission areas. Simultaneous resistance to chloroquine and antifolinics was observed in 17% of isolates between 1991 and 1994 and in 28% between 1995 and 1997.     

Molecular epidemiology of malaria in Yaoundé, Cameroon. III. Analysis of chloroquine resistance and point mutations in the multidrug resistance 1 (pfmdr 1) gene of Plasmodium falciparum

It has been postulated that chloroquine resistance may be associated with a single point mutation at codon 86 of the Plasmodium falciparum multidrug resistance 1 (pfmdr 1) gene. Using a simple and rapid molecular technique involving polymerase chain reaction and restriction fragment length polymorphism, the frequency of the Asn-to-Tyr mutation associated with chloroquine resistance was established among 129 clinical isolates obtained from indigenous patients in Yaoundé, Cameroon. The results showed that 110 of 129 isolates display a mutant codon. The other clinical isolates had either a pure wild-type Asn-86 codon (n = 12) or mixed Asn/Tyr alleles (n = 7). In vitro drug assays were performed to compare the genotype and phenotype in 102 clinical isolates. Of these isolates, 86 displayed pure Tyr-86 mutant codon; 48 (56%) mutant isolates were chloroquine-resistant (50% inhibitory concentration [IC50] > 100 nM), as expected, but 38 (44%) mutant isolates were chloroquine-sensitive (IC50 < 100 nM). Three chloroquine-resistant isolates and seven chloroquine-sensitive parasites carried a wild-type Asn-86 codon. Mixed alleles were found in six isolates (four chloroquine-sensitive and two chloroquine-resistant isolates). Our results did not confirm previous observations on the possible association between chloroquine resistance phenotype and genotype based on the pfmdr 1 gene.     

Physicochemical properties correlated with drug resistance and the reversal of drug resistance in Plasmodium falciparum

At high molar excess, verapamil can selectively increase the accumulation and cytotoxicity of structurally dissimilar natural product drugs in many multidrug-resistant tumor cell lines. Such concentrations of verapamil are also capable of increasing the accumulation and activity of chloroquine in chloroquine-resistant strains of the human malaria parasite Plasmodium falciparum. Despite such similarities, it is not clear why chloroquine-resistant P. falciparum is often susceptible to closely related compounds such as amodiaquine, whereas cancer cells are cross-resistant to many structurally unrelated drugs. For 13 aminoquinoline and aminoacridine compounds, relative drug resistance was negatively correlated with lipid solubility at physiological pH (r2 = 0.90, p < 0.0001). The ability of verapamil (5 microM) to reverse drug resistance was also negatively correlated with lipid solubility (r2 = 0.88, p < 0.0001). Furthermore, molar refractivity was weakly correlated with relative drug resistance (r2 = 0.46, p < 0.05) and reversal of drug resistance (r2 = 0.52, p < 0.005). Verapamil increases chloroquine accumulation by resistant parasites, a mechanism suggested to account for its selective chemosensitization effect. We show that the initial rate of chloroquine accumulation by resistant parasites is increased by verapamil. This effect of verapamil is abolished when deoxy-glucose is substituted for glucose. Therefore, verapamil produces an energy-dependent increase in the permeability of resistant parasites to chloroquine. For a panel of four chloroquine-resistant and two chloroquine-susceptible isolates, the effect of verapamil on the accumulation of chloroquine and monodesethyl amodiaquine was found to be correlated (r2 = 0.96, p < 0.001). Verapamil chemosensitization was also correlated for the two drugs (r2 = 0.92, p < 0.005), suggesting a common mechanism. In summary, the degree of drug resistance and the extent of verapamil chemosensitization for a particular drug seem to be dependent on general physical features such as lipid solubility and molar refractivity rather than on closely defined structural parameters. These studies provide insight into this important resistance mechanism of malaria parasites and may provide direction for the development of new drugs that are effective against resistant parasites.     

In vitro activity of artemisinin derivatives against African isolates and clones of Plasmodium falciparum

To evaluate the effect of intensive physical exercise on intraocular pressure (IOP) in 66- to 85-year-old subjects IOP was measured before and after a maximal bicycle ergometer test. The non-glaucomatous subjects comprised 85 males and 36 female athletes and 16 male and 22 female controls of corresponding age drawn from a population register. IOP was measured using a non-contact tonometer. The results indicated a decrease (> or = 2 mmHg) in 34% of the subjects, no change in 57% and an increase in 9%. The decrease was more pronounced in subjects with higher pre-test values. In all four subjects with a pre-test value above 22 mmHg a reduction from 4 to 11 mmHg was observed. The change in IOP during physical loading was not significantly associated with the intensity and duration of exercise test. Three of the 5 male subjects with diagnosed glaucoma and undergoing hypotensive medication, who were analyzed separately, also showed a reduction in IOP during loading. In the pre- or post-test values there were no differences between the athletes and controls, while women tended to have higher IOP values than men. It is concluded that physical loading has predominantly a moderating effect, if any, on IOP in elderly men and women.     

Promiscuity of clinical Plasmodium falciparum isolates for multiple adhesion molecules under flow conditions

The central pathologic process in severe Plasmodium falciparum malaria is the cytoadherence of parasitized erythrocytes to capillary and postcapillary venular endothelium, with resultant tissue hypoxia, metabolic disturbances, and multiorgan dysfunction. The molecular basis of this process has been studied extensively using static adhesion assays. In the present study, we determined whether infected red blood cells (IRBC) from clinical parasite isolates would roll and adhere on CD36, ICAM-1, E-selectin, P-selectin, and VCAM-1 using a laminar flow system that allowed for the direct visualization of IRBC-substratum interactions. The results indicate that IRBC could tether and roll on CD36, ICAM-1, P-selectin, and VCAM-1 in a shear-dependent fashion, but significant adhesion was restricted to CD36. There was no interaction with E-selectin. When both CD36 and ICAM-1 were expressed on the same cellular substratum such as C32 melanoma cells, adhesion was significantly greater than when CD36 was present alone. The adhesive interactions were different from those between leukocytes and the same adhesion molecules. Furthermore, IRBC rolling on P-selectin and VCAM-1 was not inhibitable by Abs that entirely prevented leukocyte-receptor interactions. These findings suggest that cytoadherence under physiologic conditions may be a multistep process similar to that involved in the recruitment of a number of different cell types. Further elucidation of the molecular basis of these novel interactions is crucial for the development of therapeutic interventions aimed at inhibiting or reversing the process.     

In vitro drug sensitivity and clinical aspects of Plasmodium falciparum malaria in African children

OBJECTIVE: To compare body mass index (BMI), lipid, lipoprotein and apolipoprotein concentrations according to the Hind III and Pvu II restriction polymorphisms of the LPL gene in obese subjects. DESIGN: Cross sectional study of anthropometric and lipid variables in relation to genetic factors. SETTING: Nutrition Outpatient Clinic of Bichat Hospital in Paris, France. SUBJECTS: 236 unrelated patients (162 women and 74 men) were selected on the basis of 120% of ideal body weight. MAIN OUTCOME MEASURES: Anthropometry (body mass index, waist to hip ratio), blood lipids and lipoproteins, determination of LPL Hind III and Pvu II genotypes. RESULTS: Digestion with Hind III generated two alleles, H1 (absence of cutting site) and H2 (presence of cutting site), with frequencies of 0.30 and 0.70 respectively. Digestion with Pvu II generated two alleles P1 and P2 with frequencies of 0.49 and 0.51 respectively. The Hind III polymorphism was significantly associated with body mass index (BMI) (P < 0.05). The H2H2 genotype was associated with hypertriglyceridemia: 68% of the hypertriglyceridemic subjects have the H2H2 genotype vs 43% of the normotriglyceridemic group (P < 0.05). Plasma triglyceride levels varied significantly among the Hind III genotypes, H2H2 genotype having the highest total and VLDL-triglyceride levels; the Hind III polymorphism also showed a significant association with HDL2-cholesterol. These associations were only seen in women and were not explained by the variations in BMI and age. No significant associations were found between lipid traits and Pvu II genotype. CONCLUSION: These results suggest that genetic variation in the LPL gene in obese subjects is associated with hypertriglyceridemia and possibly with a predisposition to obesity.     

In vitro activities of novel antifolate drug combinations against Plasmodium falciparum and human granulocyte CFUs

The potency of antimalarial dihydrofolate reductase inhibitors, alone and in synergistic combination with dihydropteroate synthetase inhibitors, against the Kenyan K39 strain of Plasmodium falciparum (pyrimethamine resistant) and against normal replicating human bone marrow cells in in vitro culture has been studied. Therapeutic indices and rank order of synergistic potency were derived. Trimethoprim, pyrimethamine, and the quinazolines WR159412 and WR158122 had the smallest therapeutic indices (1.39, 4.38, 2.56, and 90.0, respectively), while the three triazines clociguanil, WR99210, and chlorcycloguanil had the largest (3,562, 3,000, and 2,000, respectively). In rank order of decreasing activity against P. falciparum, the six most potent drug combinations were WR99210-dapsone, chlorcycloguanil-dapsone, WR158122-dapsone, WR159412-dapsone, WR159412-sulfamethoxazole, and chlorcycloguanil-sulfamethoxazole; pyrimethamine-sulfadoxine was the least potent combination. These experiments form a basis for the selection of rapidly eliminated antifolate combinations for further clinical testing.     

tRNA genes transcribed from the plastid-like DNA of Plasmodium falciparum

Besides their mitochondrial genome, malarial parasites contain a second organellar DNA. This 35 kb circular molecule has a number of features reminiscent of plastid DNAs. Sequence analysis shows that along with other genes the circle codes for 25 different tRNAs all of which are transcribed. Six of the tRNAs have some unusual features, and one has an intron, the only one found so far on the circle. Comparison of codon and anticodon usage indicates that the 25 tRNAs are sufficient to decode all the protein genes present on the circle. The maintenance of such a parsimonious but complete translation system is further evidence for the functionality of the circle.     

Control of gene expression in Plasmodium falciparum

Myocardial contusion is an infrequent, but sometimes serious complication in patients who experienced deceleration (blunt) trauma. We investigated the assessment of the new cardiac markers troponin I (cTnI) and troponin T (cTnT) in relation to the conventional CKMB-activity, the CKMB-activity/CK-total ratio, CKMB-mass and the CKMB-mass/CK-total ratio for the detection of myocardial contusion in 89 patients with blunt trauma (38 patients with thoracic injuries and 51 patients without thoracic injuries). All parameters were analysed at admission (t1) and 24 h after admission (t2). For the patients with thoracic injuries, at t1 cTnI was elevated in three, and cTnT in four patients; at t2 both cTnI and cTnT were elevated in nine patients. At t1, eighteen to thirty patients had increased levels of the conventional parameters; at t2 this was true for six to thirty-five patients. For the patients without thoracic injuries all cTnI and cTnT levels were within the reference ranges at t1. At t2 one patient, who experienced an acute myocardial infarction, had elevated cTnI and cTnT levels. At t1, five to thirty-five patients had increased levels of the conventional parameters; at t2 this was true for four to forty-two patients. From this study we conclude that the conventional parameters are not useful for the detection of myocardial contusion in patients experiencing blunt trauma. The parameters cTnI and cTnT are equally accurate and more reliable for the selection of patients who require intensive cardiac monitoring. If at admission the cTnI or the cTnT levels are within the reference ranges, a second analysis after admission is necessary to reach a reliable conclusion concerning myocardial contusion as a result of trauma on basis of the troponin levels.     

In vitro culture and drug sensitivity assay of Plasmodium falciparum with nonserum substitute and acute-phase sera

The short-term in vitro growth of Plasmodium falciparum parasites in the asexual erythrocytic stage and the in vitro activities of eight standard antimalarial drugs were assessed and compared by using RPMI 1640 medium supplemented with 10% nonimmune human serum, 10% autologous or homologous acute-phase serum, or 0.5% Albumax I (lipid-enriched bovine serum albumin). In general, parasite growth was maximal with autologous (or homologous) serum, followed by Albumax I and nonimmune serum. The 50% inhibitory concentrations (IC50s) varied widely, depending on the serum or serum substitute. The comparison of IC50s between assays with autologous and nonimmune sera showed that monodesethylamodiaquine, halofantrine, pyrimethamine, and cycloguanil had similar IC50s. Although the IC50s of chloroquine, monodesethylamodiaquine, and dihydroartemisinin were similar with Albumax I and autologous sera, the IC50s of all test compounds obtained with Albumax I differed considerably from the corresponding values obtained with nonimmune serum. Our results suggest that Albumax I and autologous and homologous sera from symptomatic, malaria-infected patients may be useful alternative sources of serum for in vitro culture of P. falciparum isolates in the field. However, autologous sera and Albumax I do not seem to be suitable for the standardization of isotopic in vitro assays for all antimalarial drugs.     

Receptor-specific adhesion and clinical disease in Plasmodium falciparum

Rapid detection of single-base changes is fundamental to molecular medicine. PASA (PCR Amplification of Specific Alleles) is a rapid method of genotyping single-base changes, but one reaction is required for each allele. Bidirectional PASA (Bi-PASA) was developed to distinguish between homozygotes and heterozygotes in one PCR reaction by utilizing novel primer design with appropriate cycling conditions. In Bi-PASA, one of the alleles is amplified by a PASA reaction in one direction while the second allele is amplified by a PASA reaction in the opposite direction. Two outer (P and Q) and two inner allele-specific (A and B) primers are required. In heterozygotes, three segments are amplified: a segment of size AQ resulting from one allele, another segment of size PB resulting from the second allele, and a combined segment of size PQ. In homozygotes, segment PQ and either segments AQ or PB amplify. The two inner primers (A and B) contain a relatively short complementary region and a 10-nucleotide G + C-rich 5' tail. The inner primers "switch" from low-efficiency to high-efficiency amplification when genomic DNA is replaced by previously amplified template DNA. In addition, the 5' tails prevent "megapriming". The parameters for optimizing Bi-PASA were investigated in detail for common mutations in the human factor V and catechol-O-methyltransferase genes. Guidelines for optimization of Bi-PASA also were developed and tested in a prospective study. Three additional Bi-PASA assays were optimized rapidly by utilizing these guidelines. In conclusion, Bi-PASA is a simple and rapid method for detecting the zygosity of known mutations in a single PCR reaction.     

Fluconazole susceptibility of Candida isolates from oropharyngeal candidosis

New technologies in molecular biology will allow the improvement of screening, diagnosis and prognosis of colorectal cancer patients. For example the determination of germline mutation in APC or in mismatch repair genes in patient with familial adenomatous polyposis or with HNPCC is now possible. The clinical surveillance can be restricted to the patients with these germline defects. More over the knowledge of somatic genetic alterations in colorectal cancer cells seems to be useful in the determination of prognosis of these patients or in order to predict the chemotherapy response.     

Relationship between antimalarial drug activity, accumulation, and inhibition of heme polymerization in Plasmodium falciparum in vitro

We have investigated the contribution of drug accumulation and inhibition of heme polymerization to the in vitro activities of a series of antimalarial drugs. Only those compounds exhibiting structural relatedness to the quinolines inhibited heme polymerization. We could find no direct correlation between in vitro activity against chloroquine-susceptible or chloroquine-resistant isolates and either inhibition of heme polymerization or cellular drug accumulation for the drugs studied. However, in vitro activity against a chloroquine-susceptible isolate but not a chloroquine-resistant isolate showed a significant correlation with inhibition of heme polymerization when the activity was normalized for the extent of drug accumulation. The importance of these observations to the rational design of new quinoline-type drugs and the level of agreement of these conclusions with current views on quinoline drug action and resistance are discussed.     

In vitro activity of lumefantrine (benflumetol) against clinical isolates of Plasmodium falciparum in Yaoundé, Cameroon

The in vitro antimalarial activity of the new Chinese synthetic drug, lumefantrine, also known as benflumetol (a fluorene derivative belonging to the aminoalcohol class), was determined by an isotopic microtest against 61 fresh clinical isolates of Plasmodium falciparum and compared with that of other established antimalarial agents. The geometric mean 50% inhibitory concentration of lumefantrine was 11.9 nmol/liter (95% confidence intervals, 10.4 to 13.6 nmol/liter; range, 3.3 to 25.6 nmol/liter). The in vitro activities of lumefantrine against the chloroquine-sensitive and the chloroquine-resistant isolates did not differ (P > 0.05). There was a significant positive correlation of responses between lumefantrine and two other aminoalcohols studied, mefloquine (r = 0.688) and halofantrine (r = 0.677), and between lumefantrine and artesunate (r = 0.420), suggesting a potential for in vitro cross-resistance. Our data suggest high in vitro activity of lumefantrine, comparable to that of mefloquine, and are in agreement with the promising results of preliminary clinical trials.     

Evaluation of mycobacteria growth indicator tube for recovery and drug susceptibility testing of Mycobacterium tuberculosis isolates from respiratory specimens

The new BBL mycobacteria growth indicator tube (MGIT) was evaluated for its ability to detect mycobacteria directly from patient specimens and to determine the drug susceptibility of Mycobacterium tuberculosis isolates. A total of 85 respiratory specimens were tested. Specimens were digested, concentrated, examined microscopically for acid-fast bacilli, and inoculated into MGITs and onto Lowenstein-Jensen slants by standard procedures. The tubes were incubated at 37 degrees C and were examined daily for fluorescence to 365-nm UV light. All 25 specimens smear positive for acid-fast bacilli were tested for drug susceptibility in MGITs containing 1.0 mu g of rifampin per ml, 0.1 mu g of isoniazid per ml, 2.0 mu g of streptomycin per ml, and 2.0 mu g of ofloxacin per ml. These results were compared with those obtained by testing the same M. tuberculosis isolates by the indirect proportion method at drug concentrations of 4.0 mu g of rifampin per ml, 0.2 mu g of isoniazid per ml, 2.0 mu g of ethambutol per ml. 4.0 mu g of streptomycin per ml, and 2.0 mu g of ofloxacin per ml. No significant difference in the sensitivity of detection of M. tuberculosis isolates was found between the two methods. However, the time to detection was significantly shorter in MGITs. Drug susceptibility test results for M. tuberculosis isolates by the two methods demonstrated an excellent correlation. The mean time to reporting of drug susceptibility results was 5 days for MGITs versus 16 days for Lowenstein-Jensen slants. The results of this preliminary study indicate that the MGIT system appears to have potential for routine use in mycobacteriology for both the detection and the drug susceptibility testing of M. tuberculosis isolates. However, it is important to emphasize that simple nonautomated equipment should be developed to improve the accuracy of fluorescence detection.