Fátima Nogueira

Detection of atovaquone and Malarone resistance conferring mutations in Plasmodium falciparum cytochrome b gene (cytb).

Clinical treatment failures of the hydroxynaphthoquinone atovaquone or its combination with proguanil (Malarone) in Plasmodium falciparum malaria has been recently documented. These events have been associated to single nucleotide polymorphisms (SNPs) in the parasite cytochrome b gene (cytb). In this report we describe a set of nest PCR-RFLP methods developed for the fast detection of all known cytb mutations associated to resistance to these drugs. The methods were successfully applied for the analysis of phenol-chloroform extracted DNA samples from patients not cured by Malarone, and from an established parasite clone. Further, the protocol for the detection of the A803C mutation was applied to 164 DNA field samples extracted through crude methanol-based protocols, originated from several malaria settings. The PCR-RFLP methods here presented can be used as a valuable for the clinical detection and study of Malarone and atovaquone P. falciparum resistance.

Comparative Study of Brain CD8+ T Cells Induced by Sporozoites and Those Induced by Blood-Stage Plasmodium berghei ANKA Involved in the Development of Cerebral Malaria

ABSTRACT To obtain insight into the mechanisms that contribute to the pathogenesis of Plasmodium infections, we developed an improved rodent model that mimics human malaria closely by inducing cerebral malaria (CM) through sporozoite infection. We used this model to carry out a detailed study on isolated T cells recruited from the brains of mice during the development of CM. We compared several aspects of the immune response related to the experimental model of Plasmodium berghei ANKA infection induced by sporozoites in C57BL/6 mice and those related to a blood-stage infection. Our data show that in both models, oligoclonal TCRVβ4+, TCRVβ6+, TCRVβ8.1+, and TCRVβ11+ major histocompatibility complex class I-restricted CD8 T cells were present in the brains of CM+ mice. These CD8+ T cells display an activated phenotype, do not undergo apoptosis, secrete gamma interferon or tumor necrosis factor alpha, and are associated with the development of the neurological syndrome.

Molecular characterisation of drug-resistant Plasmodium falciparum from Thailand

BackgroundThe increasing levels of Plasmodium falciparum resistance to chloroquine (CQ) in Thailand have led to the use of alternative antimalarials, which are at present also becoming ineffective. In this context, any strategies that help improve the surveillance of drug resistance, become crucial in overcoming the problem.MethodsIn the present study, we have established the in vitro sensitivity to CQ, mefloquine (MF), quinine (QUIN) and amodiaquine (AMQ) of 52 P. falciparum isolates collected in Thailand, and assessed the prevalence of four putative genetic polymorphisms of drug resistance, pfcrt K76T, pfmdr1 N86Y, pfmdr1 D1042N and pfmdr1 Y1246D, by PCR-RFLP.ResultsThe percentage of isolates resistant to CQ, MF, and AMQ was 96% (50/52), 62% (32/52), and 58% (18/31), respectively, while all parasites were found to be sensitive to QUIN. In addition, 41 (79%) of the isolates assayed were resistant simultaneously to more than one drug; 25 to CQ and MF, 9 to CQ and AMQ, and 7 to all three drugs, CQ, MF and AMQ. There were two significant associations between drug sensitivity and presence of particular molecular markers, i) CQ resistance / pfcrt 76T (P = 0.001), and ii) MF resistance / pfmdr1 86N (P < 0.001)Conclusionsi) In Thailand, the high levels of CQ pressure have led to strong selection of the pfcrt 76T polymorphism and ii) pfmdr1 86N appears to be a good predictor of in vitro MF resistance.

Plasmodium vivax Chloroquine Resistance and Anemia in the Western Brazilian Amazon

ABSTRACT Data on chloroquine (CQ)-resistant Plasmodium vivax in Latin America is limited, even with the current research efforts to sustain an efficient malaria control program in all these countries where P. vivax is endemic and where malaria still is a major public health issue. This study estimated in vivo CQ resistance in patients with uncomplicated P. vivax malaria, with use of CQ and primaquine simultaneously, in the Brazilian Amazon. Of a total of 135 enrolled subjects who accomplished the 28-day follow-up, parasitological failure was observed in 7 (5.2%) patients, in whom plasma CQ and desethylchloroquine (DCQ) concentrations were above 100 ng/dl. Univariate analysis showed that previous exposure to malaria and a higher initial mean parasitemia were associated with resistance but not with age or gender. In the multivariate analysis, only high initial parasitemia remained significant. Hemoglobin levels were similar at the beginning of the follow-up and were not associated with parasitemia. However, at day 3 and day 7, hemoglobin levels were significantly lower in patients presenting CQ resistance. The P. vivax dhfr (pvdhfr), pvmrp1, pvmdr1, and pvdhps gene mutations were not related to resistance in this small sample. P. vivax CQ resistance is already a problem in the Brazilian Amazon, which could be to some extent associated with the simultaneous report of anemia triggered by this parasite, a common complication of the disease in most of the areas of endemicity.

Prevalence of pfmdr1, pfcrt, pfdhfr and pfdhps mutations associated with drug resistance, in Luanda, Angola

BackgroundMalaria is the infectious disease causing the highest morbidity and mortality in Angola and due to widespread chloroquine (CQ) resistance, the country has recently changed its first-line treatment recommendations for uncomplicated malaria, from CQ to artemisinin combination therapies (ACT) in adults, and sulphadoxine/pyrimethamine (S/P) in pregnant women. Loss of SP sensitivity is, however, progressing rapidly in Africa and, in this study, were investigated a number of molecular markers associated to CQ and S/P.MethodsBlood samples were collected from 245 children with uncomplicated malaria, admitted at the Pediatric Hospital Dr. David Bernardino (HPDB), Angola, and the occurrence of mutations in Plasmodium falciparum was investigated in the pfmdr1 (N86Y) and pfcrt (K76T) genes, associated with CQ resistance, as well as in pfdhfr (C59R) and pfdhps (K540E), conferring SP resistance.ResultsThe frequencies of pfmdr1 mutations in codon 86 were 28.6% N, 61.3% Y and 10.1% mixed infections (NY). The frequency of pfcrt mutations in codon 76 were 93.9% K, 5.7% T and 0.4% mixed infections (KT). For pfdhfr the results were in codon 59, 60.6% C, 20.6% R and 18.8% mixed infections (CR). Concerning pfdhps, 6.3% of the isolates were bearers of the mutation 540E and 5.4% mixed infections (K540E).ConclusionThe results of this epidemiologic study showed high presence of CQ resistance markers while for SP a much lower prevalence was detected for the markers under study.

Novel Endoperoxide-Based Transmission-Blocking Antimalarials with Liver- and Blood-Schizontocidal Activities

In a search for effective compounds against both the blood- and liver-stages of infection by malaria parasites with the ability to block the transmission of the disease to mosquito vectors, a series of hybrid compounds combining either a 1,2,4-trioxane or 1,2,4,5-tetraoxane and 8-aminoquinoline moieties were synthesized and screened for their antimalarial activity. These hybrid compounds showed high potency against both exoerythrocytic and erythrocytic forms of malaria parasites, comparable to representative trioxane-based counterparts. Furthermore, they efficiently blocked the development of the sporogonic cycle in the mosquito vector. The tetraoxane-based hybrid 5, containing an amide linker between the two moieties, effectively cleared a patent blood-stage P. berghei infection in mice after i.p. administration. Overall, these results indicate that peroxide-8-aminoquinoline hybrids are excellent starting points to develop an agent that conveys all the desired antimalarial multistage activities in a single chemical entity and, as such, with the potential to be used in malaria elimination campaigns.

Probing the aurone scaffold against Plasmodium falciparum: design, synthesis and antimalarial activity.

A library comprising 44 diversely substituted aurones derivatives was synthesized by straightforward aldol condensation reactions of benzofuranones and the appropriately substituted benzaldehydes. Microwave enhanced synthesis using palladium catalyzed protocols was introduced as a powerful strategy for extending the chemical space around the aurone scaffold. Additionally, Mannich-base derivatives, containing a 7-aminomethyl-6-hydroxy substitution pattern at ring A, were also prepared. Screening against the chloroquine resistant Plasmodium falciparum W2 strain identified novel aurones with IC50 values in the low micromolar range. The most potent compounds contained a basic moiety, with the ability to accumulate in acidic digestive vacuole of the malaria parasite. However, none of those aurones revealed significant activity against hemozoin formation and falcipain-2, two validated targets expressed during the blood stage of P. falciparum infection and functional in digestive vacuole of the parasite. Overall, this study highlight (i) the usefulness of aurones as platforms for synthetic procedures using palladium catalyzed protocols to rapidly deliver lead compounds for further optimization and (ii) the potential of novel aurone derivatives as promising antimalarial compounds.

In vitro chloroquine resistance for Plasmodium vivax isolates from the Western Brazilian Amazon

BackgroundChloroquine (CQ) and primaquine (PQ) are still the drugs of choice to treat Plasmodium vivax malaria in many endemic areas, Brazil included. There is in vivo evidence for the P. vivax resistance to CQ in the Brazilian Amazon, where the increase in the proportion of P. vivax malaria parallels the increase of unusual clinical complications related to this species. In this study, in vitro CQ and mefloquine (MQ)-susceptibility of P. vivax isolates from the Western Brazilian Amazon was tested using the double-site enzyme-linked lactate dehydrogenase immunodetection (DELI) assay.MethodsA total of 112 P. vivax isolates were tested in vitro for CQ-susceptibility and out of these 47 were also tested for MQ-susceptibility. The DELI assay was used to detect P. vivax growth at 48-hour short-term culture in isolates with ring stages ranging from 50 to %. Each isolate was tested in triplicate and geometric means of IC50’s was obtained. Nineteen isolates were genetically characterized for pvdhfr, pvmrp1, pvmdr1 and pvdhps candidate genes likely related to CQ resistance (10 with IC50<40 nM and 9 with IC50 >100 nM).ResultsTwelve out of 112 isolates were considered resistant to CQ, resulting in 10.7% (IC95% 5.0-16.4), while 3 out of 47 (6.4%; IC95% 0.0-12.8) were resistant to MQ. A discrete correlation was observed between IC50’s of CQ and MQ (Spearman=0.294; p=0.045). For pvdhps gene, a non-synonymous mutation was found at codon 382 (S→C) in 5/8 CQ-sensitive samples and 1/9 CQ-resistant samples (p=0.027). The other molecular markers were not associated to CQ-susceptibility.ConclusionsIn vitro CQ-resistance estimated in this study, estimated by the DELI test, was very similar to that observed in clinical trials, suggesting that in vitro procedures developed by capable local laboratories are useful in the surveillance of CQ-resistance in the Amazon; concurrent Amazon P. vivax strains with both CQ and MQ resistance may be common; and a non-synonymous mutation at pvdhps codon 382 (S→C) was associated to in vitro susceptibility to CQ, needing further studies to be confirmed.

Epidemiological surveillance of colonising group B Streptococcus epidemiology in the Lisbon and Tagus Valley regions, Portugal (2005 to 2012): emergence of a new epidemic type IV/clonal complex 17 clone

This study presents the serotype distribution and the antibiotic resistance profile of 953 colonising group B Streptococcus (GBS) recovered from women of child bearing age (15 to 49 years) between 2005 and 2012 in the Lisbon and Tagus Valley region, Portugal. Overall, serotypes Ia, II, III, and V were the most common, accounting 752 of the 953 isolates (about 80%). However, there were changes in GBS distribution, in particular in the two last years of the study. Of note, the proportion of serotype IV isolates increased from 1% (2/148) in 2006 to 20% (19/97) in 2012. Also, considerable proportions of serotype IV isolates from 2010 to 2012 were respectively resistant to erythromycin (9/43; 21%) or clindamycin (6/43; 14%). The identification of nine serotype IV isolates presenting a novel association with the clonal complex (CC) 17 lineage, involving a putative capsular switch, may accentuate their virulence potential and ecological success. Molecular analysis of this subgroup of isolates revealed the presence of rib, IS (insertion sequence) 861 and GBSi1 group II intron within the C5a peptidase gene (scpB) – laminin-binding protein gene (lmb) region, reflecting high clonality and a putative common origin. A close surveillance of the emergent type IV/CC17 isolates is crucial considering the potential impact over GBS treatment guidelines and capsular vaccine development.

Novel potent metallocenes against liver stage malaria.

ABSTRACT Novel conjugates of the antimalarial drug primaquine (compound 1) with ferrocene, named primacenes, have been synthesized and screened for their activities against blood stage and liver stage malaria in vitro and host-vector transmission in vivo. Both transmission-blocking and blood-schizontocidal activities of the parent drug were conserved only in primacenes bearing a basic aliphatic amine group. Liver stage activity did not require this structural feature, and all metallocenes tested were comparable to or better than primaquine in this regard. Remarkably, the replacement of primaquines aliphatic chain by hexylferrocene, as in compound 7, led to a ∼45-fold-higher level activity against liver stage parasitemia than that of primaquine.