Survival patterns of Streptococcus suis serotypes 1 and 14 in porcine blood indicate cross-reactive bactericidal antibodies in naturally infected pigs.

Streptococcus suis serotype (cps) 1 and cps14 have been detected in association with severe diseases such as meningitis and polyarthritis in pigs. Though these two cps are very similar, only cps14 is an important zoonotic agent in Asia and only cps1 is described to be associated with diseases in suckling piglets rather than weaning piglets. The main objective of this study was to assess restriction of survival of cps14 and cps1 in porcine blood by IgG and IgM putatively cross-reacting with these two cps. Furthermore, we differentiate recent European cps1/14 strains by agglutination, cpsK sequencing, MLST and virulence-associated gene profiling. Our data confirmed cps1 of clonal complex 1 as an important pathotype causing polyarthritis in suckling piglets in Europe. The experimental design included also bactericidal assays with blood samples drawn at different ages of piglets naturally infected with different S. suis cps types including cps1 but not cps14. We report survival of a cps1 and a cps14 strain (both of sequence type 1) in blood of suckling piglets with high levels of maternal IgG binding to the bacterial surface. In contrast, killing of cps1 and cps14 was recorded in older piglets due to an increase of IgM as demonstrated by specific cleavage of IgM. Heterologous absorption of antibodies with cps1 or cps14 is sufficient to significantly increase the survival of the other cps. In conclusion, IgM elicited by natural S. suis infection is crucial for killing of S. suis cps1 and cps14 in older weaning piglets and has most likely the potential to cross-react between cps1 and cps14.

Gender differences in gastrointestinal disturbances and plasma concentrations of tafenoquine in healthy volunteers after tafenoquine administration for post-exposure vivax malaria prophylaxis.

In an open-label sequential cohort study, we compared gastrointestinal (GI) disturbances and plasma tafenoquine concentrations after administration of single-dose (400mg daily x 3 days; n=76 males, 11 females) and split-dose (200 mg twice daily x 3 days; n=73 males, 13 females) tafenoquine regimens in healthy Australian Defence Force volunteers for post-exposure malaria prophylaxis. The female and male volunteers had comparable demographic characteristics (age, weight, height) in the single- and split-dose treatment groups. GI disturbances were generally mild and self-limiting for both groups. The frequency of nausea and abdominal distress was over two-fold higher in females than in males for both treatment groups. Reporting of GI disturbances in the single-dose group differed significantly between males and females, but this gender difference was not seen for the split-dose group. In those volunteers who experienced GI disturbances, the mean plasma tafenoquine concentrations 12 h after the last dose of tafenoquine were approximately 1.3-fold higher in females than in males (means+/-SD: 737+/-118 ng/ml vs. 581+/-113 ng/ml). These preliminary findings suggest that further studies are required in a larger number of females to determine whether there is a need to reduce the dose of tafenoquine to minimise GI disturbances in females.

The chequered history of malaria control: are new and better tools the ultimate answer?

Following Ronald Ross' demonstration in 1897 that mosquitoes transmit malarial parasites, efforts to control malaria were naturally focussed on attacking the mosquito vector by various measures, mainly directed against the aquatic stages. Although the results were spectacular in some areas, there was a growing realisation that effective control of malaria depended on other factors, including the availability of better drugs than quinine and a greater understanding of the epidemiology of the disease under various environmental conditions. With the discovery of DDT, an all-out effort was made to eradicate malaria by attacking adult mosquitoes. Eradication was not achieved in many countries, mainly because of inadequate health infrastructures. With the emergence of chloroquine-resistant parasites, the search for more effective drug regimens intensified, various drugs and drug combinations were assessed, and methods were developed to monitor and assess degrees of resistance. Since resistance to drugs can develop very quickly, the use of drug combinations, especially those containing artemisinin derivatives, is now recommended. Insecticide-impregnated bednets have become the preferred method of vector control. Although the search for better tools must continue, the events of the past century emphasise the need to strengthen health systems to ensure that they are capable of delivering effective interventions and of assessing their effectiveness in controlling malaria.

Comparison of bioassay and high performance liquid chromatographic assay of artesunate and dihydroartemisinin in plasma.

The study was a comparison of bioassay and HPLC analysis of artesunate (ARTS) and dihydroartemisinin (DHA) in plasma. ARTS and DHA in plasma samples from patients treated with ARTS were quantified by HPLC and expressed as DHA. DHA-equivalents in the same plasma samples were measured using a standardised parasite culture technique. DHA concentrations estimated by both methods were highly correlated (bioassay=0.96 x HPLC+11.0; r2=0.92). At high concentrations (>12000 nmol/l) bioassay sometimes overestimated DHA. Bioassay of active drug in plasma correlates well with specific chemical analysis by HPLC. ARTS and DHA appear to account for the total antimalarial activity in plasma after ARTS administration.

Phenoxypropoxybiguanides, prodrugs of DHFR-inhibiting diaminotriazine antimalarials.

A total of 34 analogues of the biguanide PS-15 (5s), a prodrug of the diaminotriazine WR-99210 (8s), have been prepared. Several of them, such as 5b (PS-33) and 5m (PS-26), maintain or exceed the in vivo activity of PS-15 while not requiring the use of highly regulated starting materials. The putative diaminotriazine metabolites of these new analogues (compounds 8) have also been prepared and shown to maintain the activity against resistant P. falciparum strains. The structure-activity relationships of biguanides 5 and putative metabolites 8 are discussed.

Malaria prophylaxis/radical cure: recent experiences of the Australian Defence Force.

Since the eighties, the Australian Defence Force has deployed soldiers in malaria-endemic areas: Cambodia, Somalia, Rwanda, Bougainville, and East Timor. Currently, doxycycline is used as first line prophylactic drug and mefloquine is recommended for those who cannot tolerate the antibiotic. In 1998, the Australian Defence Force participated in the evaluation of tafenoquine for prophylaxis of both falciparum and vivax malaria in Thai soldiers. At the completion of this six-month study, 29 of 205 soldiers had come down with malaria including eight with falciparum malaria, 20 with vivax malaria, and one with mixed infection. A total of 28 of the 101 soldiers in the placebo group were infected with malaria as compared with only one of the 104 soldiers in the tafenoquine group. In 1999, another study was started on the island of Bougainville to compare the effectiveness a 3-day course of tafenoquine and a 14-day course of primaquine for radical cure of vivax malaria. At the present time, 411 soldiers have completed the study including 201 in tafenoquine arm and 210 in primaquine arm. Seven soldiers in each arm developed vivax malaria after returning to Australia. These results indicate that tafenoquine is not superior to primaquine in preventing vivax malaria. However study participants preferred the shorter course using tafenoquine and operationally it was found to be more suitable than primaquine.

Mutations in Plasmodium falciparum cytochrome b that are associated with atovaquone resistance are located at a putative drug-binding site.

Atovaquone is the major active component of the new antimalarial drug Malarone. Considerable evidence suggests that malaria parasites become resistant to atovaquone quickly if atovaquone is used as a sole agent. The mechanism by which the parasite develops resistance to atovaquone is not yet fully understood. Atovaquone has been shown to inhibit the cytochrome bc(1) (CYT bc(1)) complex of the electron transport chain of malaria parasites. Here we report point mutations in Plasmodium falciparum CYT b that are associated with atovaquone resistance. Single or double amino acid mutations were detected from parasites that originated from a cloned line and survived various concentrations of atovaquone in vitro. A single amino acid mutation was detected in parasites isolated from a recrudescent patient following atovaquone treatment. These mutations are associated with a 25- to 9,354-fold range reduction in parasite susceptibility to atovaquone. Molecular modeling showed that amino acid mutations associated with atovaquone resistance are clustered around a putative atovaquone-binding site. Mutations in these positions are consistent with a reduced binding affinity of atovaquone for malaria parasite CYT b.

Malaria in the Australian Defence Force during and after participation in the International Force in East Timor (INTERFET).

Malaria in Australian Defence Force members has been far more common in East Timor than in other recent overseas deployments. By six months after all 5,500 members of the International Force in East Timor had returned to Australia, 267 malaria infections had been reported to the Army Malaria Institute. Only 64 of those affected had their first clinical episode during their 4-5 months in East Timor, and about two-thirds of these infections were caused by Plasmodium falciparum. The remaining 212 soldiers developed their first symptoms after returning to Australia, and all but two infections were caused by P. vivax. After treatment, 44 soldiers had relapses of their vivax infections; 11 had a second relapse and two had a third relapse. These findings raise several issues about prevention and management of malaria in the ADF.

Indirect inhibition by antibiotics of nucleotide and deoxynucleotide biosynthesis in Plasmodium falciparum.

The effects of the antibiotics, doxycycline, azithromycin, ciprofloxacin and chloramphenicol, upon levels of nucleoside-5'-triphosphates (NTPs) and 2'-deoxynucleoside-5'-triphosphates (dNTPs) have been compared in the malarial parasite, Plasmodium falciparum, and in human CCRF-CEM leukemia cells. All 4 antibiotics had more severe effects upon levels of NTPs and dNTPs in P. falciparum compared with leukemia cells providing an explanation for their selective toxicity against malaria and their utility as antimalarial drugs. In bacteria, the first 3 drugs inhibit protein synthesis while ciprofloxacin inhibits topoisomerase II. The observed depletions of NTPs and dNTPs would be a secondary effect of the drug but may result in death of the parasite.

In vitro activities of the biguanide PS-15 and its metabolite, WR99210, against cycloguanil-resistant Plasmodium falciparum isolates from Thailand.

The in vitro activities of the new biguanide PS-15 and its putative active metabolite, WR99210, were determined against seven different isolates or clones of Plasmodium falciparum. The mean 50% inhibitory concentrations of PS-15 and WR99210 were 1,015 and 0.06 ng/ml, respectively. WR99210 was up to 363 times more potent than cycloguanil, the active metabolite of proguanil, against cycloguanil-resistant parasites. The pronounced activity of WR99210 against multidrug-resistant P. falciparum indicates that further studies are required to determine the value of the prodrug, PS-15, as an antimalarial agent.

Antimalaria activity of the triple combination of proguanil, atovaquone and dapsone.

The combination of proguanil and atovaquone has been shown to be more effective in curing drug-resistant infections of falciparum malaria than atovaquone or proguanil alone. Our current study sought to determine whether the antimalaria activity could be increased by adding dapsone. Plasma samples, obtained from individuals 4-72 h after proguanil-atovaquone administration, were 2-3 times more active against Plasmodium falciparum in vitro when dapsone was added to them. The enhanced activity of the combination of proguanil, atovaquone and dapsone is probably due to the combined activity of two synergistic combinations: proguanil-atovaquone and cycloguanil (metabolite of proguanil)-dapsone. These findings suggest that further studies are needed to evaluate the clinical value of the triple drug combination of proguanil, atovaquone and dapsone in the treatment of multi-drug resistant malaria.

Ex vivo antimalarial activity of proguanil combined with dapsone against cycloguanil-resistant Plasmodium falciparum isolates.

The ex vivo antimalarial activity of plasma samples obtained from 20 healthy Caucasian volunteers following daily proguanil (200 mg) plus dapsone (8 mg) for malaria chemoprophylaxis inhibited five cycloguanil-resistant Thai isolates of Plasmodium falciparum. All volunteers were phenotyped as extensive metabolisers (EMs) of proguanil. Three of the five isolates were obtained from Thai soldiers who had failed malaria prophylaxis on daily proguanil (200 mg) plus dapsone (4.0 or 12.5 mg). The Thai soldiers were also classified as EMs, but had relatively lower plasma cycloguanil concentrations compared to values reported in the literature for Caucasians and black Kenyans. Although the high level of parasite resistance to cycloguanil was the most likely explanation for the Thai soldiers failing prophylaxis on proguanil plus dapsone, their low cycloguanil concentrations may have also contributed to their lack of protection. However, in areas where parasites are more susceptible to cycloguanil, such as in certain regions of Africa, proguanil plus dapsone may still be an effective chemoprophylactic drug combination.

Plasma chloroquine concentrations in young and older malaria patients treated with chloroquine.

Plasma chloroquine (CQ) concentrations were measured by bioassay in young (0-4 years, n = 9) and older (5-60 years, n = 21) patients from Vanuatu infected with malaria following treatment with 25 mg/kg CQ over 3 days. CQ concentrations in young children tended to be lower than in older patients at days 2, 3, 4 and 7 after onset of treatment, with no drug present in two young children on day 3 and in one child on day 7. The greater difficulty experienced by young children to ingest all of their prescribed medication could have contributed to the lower CQ concentrations observed in the younger age group. The possibility that sub-therapeutic CQ concentrations are responsible for treatment failures in young children should be considered in areas where a high degree of CQ resistance has not yet been established. In such areas, the presence or prevalence of CQ-resistant infections should not be based on treatment failures observed in young children unless it can be confirmed that adequate blood CQ concentrations were achieved after treatment.

Anti-malarial drug resistance, malnutrition and socio-economic status.

To investigate the relationship between nutritional status and resistance to chloroquine treatment, we designed a nested case-control study prospective with respective to the outcome of Plasmodium falciparum-infected school children aged 5-14 years from Honiara, Solomon Islands. 38 chloroquine-resistant cases were compared with treatment-susceptible controls with respect to current nutritional status, behavioural characteristics, medical and demographic data. The overall prevalence of moderate malnutrition was 28%. The incidence rate of chloroquine resistance was 23%. Malnourished children were generally at higher risk for treatment failure than well-nourished ones (OR: 3.6; 95% CI: 1.6-8.4; P = 0.001). This effect was modified by socio-economic status. Stratified analysis by logistic regression resulted in an eightfold risk of resistance for malnourished children in the higher socio-economic category (OR: 8.1; 95% CI: 2.4-27.9; P = -.0001). In children from the lower socio-economic group, the effect of malnutrition was not significant (OR: 1.5; 95% CI: 0.4-5.6). Socio-economic differences may affect the immune status. The treatment response in children of lower socio-economic status, which are more exposed to malaria, may therefore be less affected by malnutrition.

New quinoline di-Mannich base compounds with greater antimalarial activity than chloroquine, amodiaquine, or pyronaridine.

We have compared the ex vivo antimalarial activity of 12 new quinoline di-Mannich base compounds containing the 7-dichloroquinoline or 7-trifluoromethylquinoline nucleus with amodiaquine, chloroquine, and pyronaridine using the Saimiri-bioassay model. Each compound was administered orally (30 mg/kg of body weight) to three or more noninfected Saimiri sciureus monkeys, and serum samples were collected at various times after drug administration and serially diluted with drug-free (control) serum. In vitro activity against the multidrug-resistant K1 isolate of Plasmodium falciparum was determined in serum samples by measuring the maximum inhibitory dilution at which the treated monkey serum inhibited schizont maturation in vitro. Of the 12 Mannich bases tested, 8 were associated with levels of ex vivo antimalarial activity in serum greater than those of amodiaquine, chloroquine, or pyronaridine 1 to 7 days after drug administration. Further studies were carried out with four of these compounds, and the results showed that the areas under the serum drug concentration-time curves for the four compounds were between 7- and 26-fold greater than that obtained for pyronaridine. Activity against four multidrug-resistant strains of P. falciparum was also much greater in serum samples collected from monkeys after administration of these four compounds than in serum samples collected after administration of pyronaridine or chloroquine. These findings suggest that these four quinoline Mannich base compounds possess a very marked and prolonged antimalarial activity and that further studies should be performed to determine their value as antimalarial drugs.

Disposition of proguanil in Thai patients with uncomplicated falciparum malaria.

The objective of this study was to examine the disposition of proguanil in malaria-infected Thai patients with acute uncomplicated falciparum malaria. Eleven patients were administered 500 mg of proguanil twice a day for three days (total dose = 3,000 mg). Four patients were tentatively classified as extensive metabolizers (EMs) and seven as poor metabolizers (PMs). The mean plasma clearances of proguanil for EMs and PMs were 1.31 and 1.10 L/hr/kg, respectively. The mean elimination half-life of proguanil was statistically longer in PMs than EMs (19.6 hr versus 16.1 hr; P = 0.01). Plasma clearance and elimination half-life of proguanil in the malaria patients were comparable with those reported in the literature for healthy Thai volunteers. In contrast to other ethnic groups. Thai EM patients had relatively low plasma concentrations of cycloguanil, the active metabolite of proguanil. None of the 11 patients treated with proguanil were cured of malaria and their phenotype status did not affect the treatment outcome. Although high levels of parasite resistance to cycloguanil were probably responsible for the poor response to proguanil treatment, the inability of Thai EM and PM patients to produce cycloguanil may have also contributed to the treatment outcome.

WR 238605, chloroquine, and their combinations as blood schizonticides against a chloroquine-resistant strain of Plasmodium vivax in Aotus monkeys.

The compound WR 238605 is a primaquine analog being developed by the U.S. Army as an antimalarial drug. Currently, there is no established treatment for Plasmodium vivax parasitemias that are not cured by chloroquine. This study tested WR 238605, chloroquine, and their combinations against a chloroquine-resistant strain of P. vivax (AMRU 1) in Aotus monkeys. A total dose of 3 mg/kg of WR 238605 given at a dosage of 1 mg/kg/day for three days cleared patent parasites in all eight monkeys but recrudescence of parasitemia occurred 15-25 days after initiation of treatment. A total dose of 9 mg/kg of WR 238605 over a three-day period cured all three monkeys of their infections. A total dose of 30 mg/kg of chloroquine did not clear patent infections in three monkeys, whereas a total dose of 60 mg/kg generally (two of three) cleared patent parasitemia but did not cure. Whereas total doses of 30 mg/kg of chloroquine or 3 mg/kg of WR 238605 given alone failed to cure, both drugs given in combination at these dosages cured two of three infections. These results indicate that WR 238605 may be an alternative treatment for chloroquine-resistant vivax malaria.

The activity of triple combinations of antifolate biguanides, with and without folinic acid, against plasmodium falciparum in vitro.

At least two triple combinations of biguanides, proguanil-atovaquone-dapsone and PS-15-atovaquone-dapsone, may be useful in treating drug-resistant infections of falciparum malaria. Each of these triple combinations can be considered as two synergistic combinations: proguanil-atovaquone and cycloguanil-dapsone, and PS-15-atovaquone and WR99210-dapsone, respectively. Since folinic acid might mitigate the possible side-effects produced by such drug combinations, both combinations were administered, with and without folinic acid, to 16 Saimiri sciureus monkeys. The antimalarial activity in serum samples collected from each monkey, 3, 6 and 24 h after drug administration, was then determined in vitro, against Plasmodium falciparum. The addition of folinic acid had no apparent affect on the antimalarial activity of the triple combinations tested. Such combinations may be useful against Pneumocystis carinii and Toxoplasma gondii in immunocompromised patients, as well as against malaria.

Effects of dual combinations of antifolates with atovaquone or dapsone on nucleotide levels in Plasmodium falciparum.

The triazine antifolates, cycloguanil and 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-[(2,4,5-trichlorophenoxy)propy loxy]-1,3,5-triazine hydrobromide (WR99210), and their parent biguanide compounds, proguanil and N-[3-(2,4,5-trichlorophenoxy)propyloxy]-n-(1-methylethyl)-imido dicarbonimidic-diamine hydrochloride (PS-15), were tested in combination with a series of antimalarial drugs for synergism against Plasmodium falciparum growing in erythrocytic culture. Four synergistic combinations were found: cycloguanil dapsone, WR99210-dapsone, proguanil-atovaquone, and PS-15-atovaquone. Cycloguanil-dapsone or WR99210-dapsone had a profound suppressive effect on the concentration of dTTP in parasites while that of dATP increased. Depletion of dTTP is consistent with cycloguanil or WR99210 inhibiting dihydrofolate reductase and dapsone inhibiting dihydropteroate synthase. For the combinations proguanil-atovaquone and PS-15-atovaquone, the levels of nucleoside triphosphates (NTPs) and dNTPs were generally suppressed, suggesting that inhibition is not through nucleotide pathways but probably through another metabolic mechanism(s). Combinations of two synergistic pairs of antimalarial drugs, (proguanil-atovaquone)-(cycloguanil-dapsone) and (PS-15-atovaquone)-(WR99210-dapsone), were tested, and it was found that NTPs and dNTPs decreased much more than for a single synergistic combination. Dual synergistic combinations could play an important role in the therapy of multidrug-resistant malaria, just as combination chemotherapy is used to treat cancer.