Phylogeny, evolutionary trends and classification of the Spathelia-Ptaeroxylon clade: morphological and molecular insights.

BACKGROUND AND AIMS: The Spathelia-Ptaeroxylon clade is a group of morphologically diverse plants that have been classified together as a result of molecular phylogenetic studies. The clade is currently included in Rutaceae and recognized at a subfamilial level (Spathelioideae) despite the fact that most of its genera have traditionally been associated with other families and that there are no obvious morphological synapomorphies for the clade. The aim of the present study is to construct phylogenetic trees for the Spathelia-Ptaeroxylon clade and to investigate anatomical characters in order to decide whether it should be kept in Rutaceae or recognized at the familial level. Anatomical characters were plotted on a cladogram to help explain character evolution within the group. Moreover, phylogenetic relationships and generic limits within the clade are also addressed. METHODS: A species-level phylogenetic analysis of the Spathelia-Ptaeroxylon clade based on five plastid DNA regions (rbcL, atpB, trnL-trnF, rps16 and psbA-trnH) was conducted using Bayesian, maximum parsimony and maximum likelihood methods. Leaf and seed anatomical characters of all genera were (re)investigated by light and scanning electron microscopy. KEY RESULTS: With the exception of Spathelia, all genera of the Spathelila-Ptaeroxylon clade are monophyletic. The typical leaf and seed anatomical characters of Rutaceae were found. Further, the presence of oil cells in the leaves provides a possible synapomorphy for the clade. CONCLUSIONS: The Spathelia-Ptaeroxylon clade is well placed in Rutaceae and it is reasonable to unite the genera into one subfamily (Spathelioideae). We propose a new tribal classification of Spathelioideae. A narrow circumscription of Spathelia is established to make the genus monophyletic, and Sohnreyia is resurrected to accommodate the South American species of Spathelia. The most recent common ancestor of Spathelioideae probably had leaves with secretory cavities and oil cells, haplostemonous flowers with appendaged staminal filaments, and a tracheidal tegmen.

In silico characterisation of putative Plasmodium falciparum vaccine candidates in African malaria populations.

Genetic diversity of surface exposed and stage specific Plasmodium falciparum immunogenic proteins pose a major roadblock to developing an effective malaria vaccine with broad and long-lasting immunity. We conducted a prospective genetic analysis of candidate antigens (msp1, ama1, rh5, eba175, glurp, celtos, csp, lsa3, Pfsea, trap, conserved chrom3, hyp9, hyp10, phistb, surfin8.2, and surfin14.1) for malaria vaccine development on 2375 P. falciparum sequences from 16 African countries. We described signatures of balancing selection inferred from positive values of Tajima's D for all antigens across all populations except for glurp. This could be as a result of immune selection on these antigens as positive Tajima's D values mapped to regions with putative immune epitopes. A less diverse phistb antigen was characterised with a transmembrane domain, glycophosphatidyl anchors between the N and C- terminals, and surface epitopes that could be targets of immune recognition. This study demonstrates the value of population genetic and immunoinformatic analysis for identifying and characterising new putative vaccine candidates towards improving strain transcending immunity, and vaccine efficacy across all endemic populations.

Low antiplasmodial activity of alkaloids and amides from the stem bark of Zanthoxylum rubescens (Rutaceae).

The stem bark of Zanthoxylum rubescens (syn. Fagara rubescens) is used for treating fevers associated with malaria in the Ivory Coast. Three alkaloids: N-nornitidine, 7,9-dimethoxy-2,3-methylenedioxybenzophenanthridine, and bis[6-5,6-dihydrochelerythrinyl)] ether; and two amides: zanthomamide and lemairamide, were isolated from the stem bark of this plant. These compounds were screened in vitro against the chloroquine-sensitive 3D7 strain and the chloroquine-resistant FCM29 strain of P. falciparum. N-nornitidine was found to be inactive. 7,9-dimethoxy-2,3-methylenedioxybenzophenanthridine, lemairamide and zanthomamide showed weak activity with average IC50 values ranging from 45.6 microM to 149.9 microM. Bis[6-15,6-dihydrochelerythrinyl)] ether was the most active of the tested compounds with mean IC50s of 14.9 +/- 1.4 microM in FCM29 strain and 15.3 +/- 3.4 microM in 3D7 strain (approximately 58 to approximately 1130 times less active than chloroquine respectively). The anti-Plasmodium activities of the tested alkaloids of Z. rubescens were low; and do not encourage the use of this plant as antimalarial.

[Surveillance of falciparum malaria susceptibility to antimalarial drugs and policy change in the Comoros]. / Surveillance de la chimiosensibilite du paludisme do à Plasmodium falciparum et changement de politique dans l'Union des Comores.

Between May and June 2001, efficacy of chloroquine was assessed in 5 sentinel sites in the 3 Comoro islands. Among the 183 children, age range between 6 and 59 months, followed up for 14 days, clinical failure rates ranged between 31.2 and 73.1% and the total failure rate (clinical and parasitological) between 50 and 88.5%. Failures were mainly early treatment failures. The Ministry of health, during a consensus meeting decided to change the first line drug and to gather baseline information on the efficacy and the tolerance of the combination artemether-lumefantrine. Between June and September 2004, among the 164 children, age range between 6 and 59 months included, the success rate of the combination was 99.4% in the 3 sites with a follow-up of 28 days. No serious drug related adverse event was reported.

[French language training course: malaria workshop organized by Institut Pasteur de Madagascar]. / Formation francophone Sud/Sud: l'atelier paludisme de l'Institut Pasteur de Madagascar.

The Malaria Workshop organized by Institut Pasteur de Madagascar is an original course that applies innovative concepts to training of health professionals involved in malaria control in endemic countries. Course objectives are to enhance the skills needed to fight malaria (transversal competencies, critical approach, and position statement), to reinforce project cycle management proficiency, and to demonstrate how the Internet can be used as a source of documentation to compensate for geographical isolation. The Malaria Workshop is a six-consecutive-week full-day course that has been presented once a year since 2003. Seventy-six researchers, physicians or health ministry officials have already benefited from this training. Teaching methods emphasize andragogy that facilitates a learner/mentor relationship promoting exchange rather than transmission of knowledge and problem-based learning that engages learners to take an active part in gathering information. These methods in combination with the diverse backgrounds and experience of course participants foster a positive dynamic environment for learning that is monitored by weekly progress evaluation. Follow-up surveys have confirmed the positive effect of this training on the professional performance of former participants who become more involved in program development and fund-raising efforts. A professional network is growing and learners are starting to their experience. In this report workshop organizers describe the course's origins and concepts and present the conclusions drawn based on the first five yearly sessions.

Evaluation of the OnSite malaria rapid test performance in Miandrivazo, Madagascar.

The performance of the malaria rapid diagnostic test OnSite-for detecting pan specific pLDH and Plasmodium falciparum specific HRP2 - was assessed during the malaria transmission peak period in Miandrivazo, in the southwestern part of Madagascar from April 20 to May 6, 2010. At the laboratory, the quality control OnSite Malaria Rapid Test according to the WHO/TDR/FIND method demonstrated that the test had good sensitivity. Of the 218 OnSite tests performed at the Miandrivazo Primary Health Center on patients with fever or a recent history of fever, four (1.8%, 95% CI: 0.6-4.9%) were invalid. Ninety four (43,1%) cases of malaria were confirmed by microscopy, of which 90 were P. falciparum malaria and 4 Plasmodium vivax malaria. With a Cohen's kappa coefficient of 0.94, the agreement between microscopy and OnSite is excellent. Compared with the rapid test CareStart™ commonly used within the public health structures in Madagascar, the sensitivity and specificity of the OnSite test were 97.9% and 96.8%.

Investigations of the Malagasy species Tachiadenus longiflorus Grisebach (Gentianaceae): linking chemical finding and traditional usage.

A decoction of the aerial parts of Tachiadenus longiflorus, a Gentianaceae endemic to Madagascar, is drunk to relieve stomach pains and dyspepsia; while a leaf decoction is drunk as a purgative or to relieve gall bladder ailments in folk medicine in Madagascar. The stem and bark have yielded a large amount of the triterpenoid, oleanolic acid, and the coumarins, scoparone and scopoletin. These compounds have been isolated previously from other sources and have shown broad pharmacological properties. We report the possible link between the compounds isolated and the traditional use of Tachiadenus longiflorus.

[Diagnosis of malaria in Antananarivo City: examination of the results obtained at the Institut Pasteur de Madagascar from 2001 to 2004]. / Diagnostic du paludisme dans la ville d'Antananarivo: réflexion à partir des résultats obtenus à l'Institut Pasteur de Madagascar de 2001 à 2004.

Malaria diagnosis is part of the daily activities of the Clinical Biology Center (CBC) of the Institut Pasteur de Madagascar in Antananarivo. Over a period of four years (2001-2004), regardless the methods being used, out of 6537 blood samples examined, 159 (2.43%) tests were positive. All four species of Plasmodium infecting human. were detected with a high prevalence of P. falciparum (87.2%). 49/159 patients were foreigners, but their files did not allow us to distinguish imported from locally acquired malaria cases. Also, among Malagasy patients, there was no possibility to recognize introduced malaria cases (contracted in coastal areas). In Madagascar malaria remains a public health problem. But fever and recent history of fever are often considered and treated as malaria. Our results demonstrated that confirmed malaria rate was very low. Reporting malaria on the basis of clinical signs overestimates malaria cases at the national level. The importance of malaria biological diagnosis is discussed in this article.

Isradipine--a calcium channel blocker--does not potentiate chloroquine antiplasmodial activity against Plasmodium falciparum.

Culturing fresh clinical isolates of P. falciparum and using the isotopic method, we tested separately chloroquine and isradipine--a calcium channel blocker--, and also the combination isradipine plus chloroquine. Tested wild isolates were chloroquine-sensitive. With regard to the combination isradipine/chloroquine, the isobolograms obtained indicate that isradipine antagonises chloroquine antiplasmodial activity. Taking into account these findings, we discuss the issues related to the calcium channel blocker molecules.

Limonoid derivatives from Astrotrichilia voamatata.

The stem bark of Astrotrichilia voamatata (Meliaceae) has yielded the novel limonoids voamatins C and D. These compounds represent a new type of pentanortriterpenoid and are unique in containing a ring A cyclic ether.

In vitro sensitivity of Plasmodium falciparum to amodiaquine compared with other major antimalarials in Madagascar.

Chloroquine has been used in Madagascar since 1945 and remains the first-line treatment for uncomplicated cases of malaria. Low-grades of resistance type R1 and R2 have been reported. Thus, in vitro tests were performed in order to monitor the drug sensitivity of Plasmodium falciparum from different study sites, with the aim of identifying alternatives to chloroquine. Chloroquine IC50 values ranged from 0.2 nM to 283.4 nM (n = 190, mean IC50 = 52.6 nM; 95% CI = 46.1-59.1 nM). Fifteen isolates (7.9%) were chloroquine-resistant. One mefloquine-resistant isolate was detected (1/139). The test isolates were sensitive to amodiaquine (n = 118), quinine (n = 212), pyrimethamine (n = 86) and cycloguanil (n = 79). The median IC50 for amodiaquine was 12.3 nM (mean IC50 = 15.3 nM, 95% CI = 13.3-17.3 nM). Amodiaquine was 3.4 times as active as chloroquine in vitro and 7 times as active as quinine against P. falciparum. These results indicate that amodiaquine may be a potent alternative to chloroquine in Madagascar. There was positive correlation between tested quinoline-containing drugs activities, which suggests in vitro cross-susceptibility.

In-vitro sensitivity of Plasmodium falciparum to chloroquine, halofantrine, mefloquine and quinine in Madagascar.

OBJECTIVE: To determine how sensitive Plasmodium falciparum is to the major antimalarial drugs in Madagascar. DESIGN: Assessment of Plasmodium falciparum isolates sensitivity to antimalarials, by use of the in-vitro radioisotope method. SETTING: Ankazobe and Saharevo in the foothill areas; and Toamasina and Tolagnaro in the coastal areas (between January 1998 and November 1999). SUBJECTS: Primary Plasmodium falciparum isolates from patients with uncomplicated malaria attack. RESULTS: Between January 1998 and November 1999, of the 293 in-vitro tests done with at least one antimalarial, 70% (205/293) were interpretable. As there was no significant difference between results from the four study sites, the data have been expressed as a whole. All of the successfully tested isolates were sensitive to halofantrine (n = 56) and to quinine (n = 199), 5.8% (12/205) of the isolates were resistant to chloroquine and 2% (4/199) to mefloquine. The geometric mean IC50 was 0.3 microg/L for halofantrine (95% CI = 0.1-0.4 microg/L); 9.4 microg/L for chloroquine (95% CI = 7.3-10.8 microg/L); 3.8 microg/L for mefloquine (95% CI = 3.3-4.3 microg/L); and 26.8 microg/L for quinine (95% CI = 24.3-29.4 microg/L). The low positive correlation found between halofantrine and chloroquine IC50s (n = 56; r = 0.41, P = 0.002) suggests a risk of cross-resistance between these two drugs. CONCLUSION: The degree and frequency of chloroquine resistance in-vitro is stationary in Madagascar compared to previous results during the last decade. The in-vitro sensitivity of P. falciparum to quinine, mefloquine and halofantrine encourages the use of these drugs as alternative in case of chloroquine treatment failure. Nevertheless, it is important to maintain and to extend malaria and drug sensitivity surveillance in Madagascar.

Monitoring the drug-sensitivity of Plasmodium falciparum in coastal towns in Madagascar by use of in vitro chemosensitivity and mutation detection tests.

The dissemination of mutant and resistant strains of Plasmodium falciparum makes a considerable contribution to the spread of drug-resistant malaria. Populations around harbours and airports could be particularly exposed to Plasmodium isolates introduced with imported cases of malaria. The use of chloroquine as well as the use of and sulfadoxine/pyrimethamine is currently an effective method for treating uncomplicated cases of malaria in Madagascar. As part of a monitoring programme, in vitro methods were used to assess the sensitivity of P. falciparum isolates in two coastal towns in Madagascar: Mahajanga on the west coast and Toamasina on the east coast. All of the isolates from both sites were sensitive to amodiaquine, quinine, pyrimethamine and cycloguanil. All of the isolates from Mahajanga were sensitive to chloroquine (n = 25; mean IC50 = 22.6 nM, 95% confidence interval: 16.8-28.7 nM), whereas three of the isolates from Toamasina were resistant to chloroquine (n = 18; mean IC50 = 66.3 nM; 95% confidence interval: 42.6-90 nM). The frequency of the Pfcrt Thr-76 and the dhfr Asn-108 mutations was estimated by PCR/RFLP. The 43 P. falciparum isolates examined, including the three in vitro chloroquine-resistant isolates from Toamasina were all wild-type (Lys-76). Phenotyping and genotyping studies suggested that the prevalence of chloroquine- and pyrimethamine-resistant isolates and of mutant strains of P. falciparum is very low. These results showed that in vitro test and genotyping of resistance markers approaches could be successfully used to monitor the emergence of drug-resistant malaria and to try to alleviate the lack of medical teams able to carry out in vivo test. The possible hazard/risk associated with imported cases of malaria is discussed.

Plasmodium falciparum resistant to chloroquine and to pyrimethamine in Comoros.

We report the outcome of chloroquine treatment and the prevalence of mutations at codon 86 of the pfmdr1 gene, at codon 76 of the pfcrt gene, and at codon 108 of the pfdhfr gene in clinical isolates of Plasmodium falciparum collected from 30 children under 10 years of age living in the Comoros Union. This in vivo study was carried out in February and March 2001 in Moroni. Chloroquine treatment failed in 23 children (76.6%; 95% confidence interval: 57.7 to 90.1%). Subsequent genotyping showed that all P. falciparum isolates (100%) harboured a tyrosine residue at position 86 in pfMDR1. 83.3% (25/30) of these isolates harboured a mutation at position 76 in pfCRT and half (15/30) of these isolates also harboured a mutation at position 108 in pfDHFR. Chloroquine resistance is a real concern in the Comoros Union. The prevalence of pfDHFR mutant parasites is alarming. The alternative drugs proposed as a replacement for chloroquine as first-line treatment in Comoros, and the strategy to monitor the drug susceptibility of Plasmodium sp in this part of the Indian Ocean sub-region are discussed.

Efficacy of artemether-lumefantrine treatment in patients with acute uncomplicated Falciparum malaria in Mayotte, a French collectivity of the Comoros Archipelago.

Mayotte is a French island located in the Comoros archipelago in the Indian Ocean. Due to the high level of resistance to chloroquine and sulfadoxine-pyrimethamine in this area, new therapeutic strategies are required. The aim was to assess and to document the efficacy of artemether-lumefantrine (AL) combination in four oral dosages. The follow-up was carried out during 21 days to monitor the antimalarial drug efficacy in an open trial in April-May, 2002. Results were obtained from 51 patients, aged from three to 46 years (12% less than five years). No case of therapeutic failure was observed. At day 2 after treatment, all the patients were apyretic and none of them had parasitaemia until day 21. This first therapeutic trial of the AL combination in the Indian Ocean sub-region shows that this association is safe, effective and rapid. AL should be an alternative treatment of uncomplicated malaria attacks in Comoros Archipelago, and will be of help to manage imported chloroquine-resistant falciparum malaria strains in Madagascar.

[Drug resistance of Plasmodium falciparum in coastal regions of Madagascar]. / Chimiorésistance de Plasmodium falciparum sur les regions côtierès malgaches.

Chloroquine is still the drug of choice for first-line treatment of uncomplicated malaria in Madagascar. However development and spread of chloroquine-resistance could compromise this therapeutic strategy in the future. The purpose of this 1997 study was to compare the efficacy of combined treatment using sulfadoxine and pyrimethamine and single-agent treatment using chloroquine for management of uncomplicated malaria. Study data were collected at four sites in coastal areas of Madagascar where transmission of malaria is perennial. Prevalence of malaria ranged from 15 p. 100 to 22 p. 100 in school children and from 24 p. 100 to 72 p. 100 in outpatient consulting spontaneously at community health centers. All four Plasmodium species affecting man were identified. Plasmodium falciparum was involved in 83 p. 100 of cases. In vivo testing of the susceptibility of Plasmodium falciparum to chloroquine was performed in 149 patients according to the standard simplified 7-day protocol of the WHO. The 35 tests in school children demonstrated no evidence of resistance. However type R1 + R2 resistance was noted in 17 of the 114 tests performed on outpatients, i.e. 14.9 p. 100. In vitro testing demonstrated chloroquine resistance in four of the 90 specimens tested, i.e. 4.4 p. 100. With regard to combined sulfadoxine/pyrimethamine treatment, 45 of 46 in vivo tests in outpatients showed no evidence of resistance. Combination treatment was more effective than single-agent treatment (p = 0.02) and could offer an effective alternative for future use.

The structural elucidation of a novel iridoid derivative from Tachiadenus longiflorus (Gentianaceae) using the LSD programme and quantum chemical computations.

Oleanolic acid, scoparone, scopoletin and a novel iridoid derivative, angelone, were isolated from Tachiadenus longiflorus (Gentianaceae). The structure of angelone was determined from NMR data, given as input to the Logic for Structure Determination Programme, and was finally confirmed by comparison of experimental 13C-NMR chemical shifts with those obtained by quantum mechanical calculations.

[Effect of the supernatant from mice liver cell primary culture on the in vitro growth of Plasmodium falciparum wild isolates]. / Effet du surnageant de culture primaire d'hépatocytes de souris sur la prolifération in vitro des isolats sauvages de Plasmodium falciparum.

Cultivation of Plasmodium falciparum has been a major research success, leading to a greater understanding of the parasite. Despite the fact that several P. falciparum clones have been maintained in continuous culture in different laboratories, research in genomics and proteomics would require parasitic material produced from fresh wild isolates. We have tested the effect of the supernatant from primary culture of mice hepatocytes on in vitro growth of P. falciparum isolates. Parasitized blood samples were collected from Madagascan malarious patients naturally infected. Isolates proliferation was assessed by use of isotopic method. The asexual erythrocytic stages of P. falciparum were grown for 42 hours in RPMI 1640-based medium plus L15 medium-based supernatant from mice liver cells culture, and in standard RPMI 1640-based medium alone. The mean of parasite growth was 1.5 times greater when the standard medium was enriched with the liver cells layer supernatant at a proportion of 10% and 15% (v/v). The usefulness of P. falciparum ex-vivo culture and of the hepatocytes in vitro primary culture is discussed.

[National Network study to perpetuate the surveillance of Plasmodium falciparum sensitivity to antimalarials in Madagascar]. / Réseau d'Etude de la Résistance (RER) pour pérenniser la surveillance de la sensibilité de Plasmodium falciparum aux antipaludiques à Madagascar.

To redefine strategy and policy to cure or to prevent malaria, there is a need to get relevant and updated data on Plasmodium sp sensitivity level to antimalarial drugs. Thus, in September 1999, the Madagascan Ministry of Health and the Institut Pasteur de Madagascar (IPM) formed a network named RER for malaria resistance surveillance. To alleviate the lack of experienced medical teams within the health centres, and due to technical and logistic matters, as part of the network activities, it was decided to give a start with the in vitro studies which are carried out at IPM. In vitro sensitivity testing is done by use of the isotopic method. Results from the study done in 2001 demonstrate that the Madagascan P. falciparum isolates are susceptible to amodiaquine (n = 215), to cycloguanil (n = 56), to pyrimethamine (n = 98) and to quinine (n = 214). One isolate (1/110 i.e. 0.9%) of mefloquine-resistant phenotype is detected from the Eastern region. P. falciparum susceptibility to chloroquine is satisfactory with 95.4% (206/216) of in vitro sensitive isolates. RER arises from the partnership and collaboration between the Madagascan Ministry of Health and the IPM. The network set-up is presented. The usefulness of the in vivo approach, and the in vitro investigations (chemosusceptibility test and screening of mutations accounting for resistance to chloroquine) to monitor the emergence and the dissemination of drug-resistant parasites in Madagascar as well as in the subregion of the Indian Ocean is discussed.