BackgroundAlthough malaria cases have substantially decreased in Southeast Brazil, a significant increase in the number of Plasmodium vivax-like autochthonous human cases has been reported in remote areas of the Atlantic Forest in the past few decades in Rio de Janeiro (RJ) state, including an outbreak during 2015–2016. The singular clinical and epidemiological aspects in several human cases, and collectively with molecular and genetic data, revealed that they were due to the non-human primate (NHP) parasite Plasmodium simium; however, the understanding of the autochthonous malarial epidemiology in Southeast Brazil can only be acquired by assessing the circulation of NHP Plasmodium in the foci and determining its hosts.MethodologyA large sampling effort was carried out in the Atlantic forest of RJ and its bordering states (Minas Gerais, São Paulo, Espírito Santo) for collecting and examining free-living NHPs. Blood and/or viscera were analyzed for Plasmodium infections via molecular and microscopic techniques.Principal findingsIn total, 146 NHPs of six species, from 30 counties in four states, were tested, of which majority were collected from RJ. Howler monkeys (Alouatta clamitans) were the only species found infected. In RJ, 26% of these monkeys tested positive, of which 17% were found to be infected with P. simium. Importantly, specific single nucleotide polymorphisms–the only available genetic markers that differentiate P. simium from P. vivax–were detected in all P. simium infected A. clamitans despite their geographical origin of malarial foci. Interestingly, 71% of P. simium infected NHPs were from the coastal slope of a mountain chain (Serra do Mar), where majority of the human cases were found. Plasmodium brasilianum/malariae was initially detected in 14% and 25% free-living howler monkeys in RJ and in the Espírito Santo (ES) state, respectively. Moreover, the malarial pigment was detected in the spleen fragments of 50% of a subsample comprising dead howler monkeys in both RJ and ES. All NHPs were negative for Plasmodium falciparum.Conclusions/SignificanceOur data indicate that howler monkeys act as the main reservoir for the Atlantic forest human malarial parasites in RJ and other sites in Southeast Brazil and reinforce its zoonotic characteristics.

Background: The diphtheria toxoid antigen is a major component in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. Although antibodies are considered critical for diphtheria protection, little is known about the antigenic determinants that maintain humoral immunity. Methods: One-hundred and twelve 15 mer peptides covering the entire sequence of diphtheria toxin (DTx) protein were prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice immunized with a triple DTP vaccine allowed mapping of continuous B-cell epitopes, topological studies, multiantigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Results: Twenty epitopes were identified, with two being in the signal peptide, five in the catalytic domain (CD), seven in the HBFT domain, and five in the receptor-binding domain (RBD). Two 17 mer (CB/Tx-2/12 and CB/DTx-4–13) derived biepitope peptides linked by a Gly-Gly spacer were chemically synthesized. The peptides were used as antigens to coat ELISA plates and assayed with human (huVS) and mice vaccinated sera (miVS) for in vitro diagnosis of diphtheria. The assay proved to be highly sensitive (99.96%) and specific (100%) for huVS and miVS and, when compared with a commercial ELISA test, demonstrated a high performance. Conclusions: Our work displayed the complete picture of the linear B cell IgG response epitope of the DTx responsible for the protective effect and demonstrated sufficient specificity and eligibility for phase IIB studies of some epitopes to develop new and fast diagnostic assays.

BackgroundMalaria is a major parasitic disease, affecting millions of people in endemic areas. Plasmodium falciparum parasites are responsible for the most severe cases and its resistance to anti-malarial drugs is notorious. This is a possible obstacle to the effectiveness of intermittent preventive treatment (IPT) based on sulfadoxine–pyrimethamine (SP) cures administrated to pregnant women (IPTp) during their pregnancy. As this intervention is recommended in Angola since 2006, it has assessed, in this country, the molecular profiles in P. falciparumdhfr and dhps, two polymorphic genes associated to pyrimethamine and sulfadoxine resistance, respectively.MethodsBlood samples from 52 falciparum patients were collected in Lubango, Angola and pfdhfr and pfdhps polymorphisms were analysed using nested-PCR and DNA sequencing.ResultsIn the pfdhfr gene, the 108N mutation was almost fixed (98 %), followed by 59R (63 %), 51I (46 %), 50R and 164L (2 %, respectively). No 16V/S mutations were found. The most common double mutant genotype was CNRN (59 + 108; 46 %), followed by CICN (51 + 108; 29 %) whereas IRN (51 + 59 + 108; 15 %), CNRNVL (59 + 108 + 164; 2 %) and RICN (50 + 51 + 108; 2 %) triple mutant genotypes were detected. Investigations of the pfdhps gene showed that the 437G mutation was the most prevalent (97 %). Only two and one samples disclosed the 540E (7 %) and the 436A (3 %), respectively. Single mutant SGKAA (437; 86 %) was higher than SGEAA (437 + 540; 7 %) or AGKAA (436 + 437; 3 %) double mutants genotypes. No polymorphism was detected at codons 581G and 613T/S. Combining pfdhfr and pfdhps alleles two triple mutant haplotypes (double mutant in dhfr and single mutant in dhps) were observed: the ACICNVI/SGKAA in 14 (56 %) samples and the ACNRNVI/SGKAA in five (20 %) samples. One quadruple mutant haplotype was detected (ACIRNVI/SGKAA) in six (24 %) P. falciparum samples. No quintuple pfdhfr–pfdhps mutant was noted.Conclusionpfdhfr and pfdhps gene mutations in isolates from Lubango are suggestive of a low-grade SP resistance and IPT for pregnant women and infant based on SP treatment could be effective. Routine molecular studies targeting polymorphism in these two genes need to be routinely conducted at country level.

Several questions regarding the production and functioning of autoantibodies (AAb) during malaria infection remain open. Here we provide an overview of studies conducted in our laboratory that shed some light on the questions of whether antiphospholipid antibodies (aPL) and other AAb associated with autoimmune diseases (AID) can recognize Plasmodia antigens and exert anti-parasite activity; and whether anti-parasite phospholipid antibodies, produced in response to malaria, can inhibit phospholipid-induced inflammatory responses and protect against the pathogenesis of severe malaria. Our work showed that sera from patients with AID containing AAb against dsDNA, ssDNA, nuclear antigens (ANA), actin, cardiolipin (aCL) and erythrocyte membrane antigens recognize plasmodial antigens and can, similarly to monoclonal AAb of several specificities including phospholipid, inhibit the growth of P. falciparum in vitro. However, we did not detect a relationship between the presence of anti-glycosylphosphatidylinositol (GPI) antibodies in the serum and asymptomatic malaria infection, although we did register a relationship between these antibodies and parasitemia levels in infected individuals. Taken together, these results indicate that autoimmune responses mediated by AAb of different specificities, including phospholipid, may have anti-plasmodial activity and protect against malaria, although it is not clear whether anti-parasite phospholipid antibodies can mediate the same effect. The potential effect of anti-parasite phospholipid antibodies in malarious patients that are prone to the development of systemic lupus erythematosus or antiphospholipid syndrome, as well as the (possibly protective?) role of the (pathogenic) aPL on the malaria symptomatology and severity in these individuals, remain open questions.

BackgroundPlasmodium vivax is the most widely distributed species causing the highest number of malaria cases in the world. In Brazil, P. vivax is responsible for approximately 84 % of reported cases. In the absence of a vaccine, control strategies are based on the management of cases through rapid diagnosis and adequate treatment, in addition to vector control measures. The approaches used to investigate P. vivax resistance to chloroquine (CQ) were exclusively in vivo studies because of the difficulty in keeping parasites in continuous in vitro culture. In view of the limitations related to follow-up of patients and to assessing the plasma dosage of CQ and its metabolites, an alternative approach to monitor chemo-resistance (QR) is to use molecular markers. Single nucleotide polymorphisms (SNPs) in the multidrug resistance gene pvmdr1 are putative determinants of CQ resistance (CQR), but such SNPs in P. vivax isolates from patients with good response to treatment should be further explored. The aim of this study is to investigate the mutations in the gene, supposedly associated to QR, in P. vivax isolates from successfully cured patients, living in Brazilian endemic and non-endemic areas.MethodsBlood samples were collected from 49 vivax malaria patients from endemic (Amazon Basin: 45) and non-endemic (Atlantic Forest: four) Brazilian regions and analysed for SNPs in the CQR-related P. vivax gene (pvmdr1), using PCR-based methods.ResultsAmong the 49 isolates genetically characterized for the gene pvmdr1, 34 (70 %) presented at least one mutation. T958M mutant alleles were the most frequent (73 %) followed Y976F (15 %) and F1076L (12 %). Single mutation was detected in 24 (70.5 %) isolates and double mutations in ten (29.5 %). The most common single mutant genotype was the 958M/Y976/F1076 (79 %), followed by 976F/F1076 (21 %) whereas 958M/Y976/1076L (60 %) and 976F/1076L (40 %) double mutant genotypes were detected. Single mutant profile was observed only in isolates from Amazon Basin, although double mutants were found both in the Amazon and Atlantic Forest regions. Interestingly, the genotype 958M/Y976/1076L was present in all isolates from the Atlantic Forest in the Rio de Janeiro State.ConclusionsConsidering that primaquine (PQ) efficacy is highly dependent on concurrent administration of a blood schizontocidal agent and that PQ could not circumvent CQR, together with the fact that no pvmdr1 mutation should be expected in successfully cured patients, these findings seem to indicate that the pvmdr1 gene is not a reliable marker of CQR. Further investigations are needed to define a reliable molecular marker for monitoring P. vivax CQR in P. vivax populations.