Cor Jf Fontes

Low sensitivity of nested PCR using Plasmodium DNA extracted from stained thick blood smears: an epidemiological retrospective study among subjects with low parasitaemia in an endemic area of the Brazilian Amazon region.

BackgroundThe success of PCR technique depends on many factors, such as high quality DNA pellets obtained from blood samples, good reagents and adequate conditions of amplification. Taking these limitations into account, a retrospective epidemiological study for malaria diagnosis was conducted in a mesoendemic area in the Brazilian Amazon.MethodsA nested PCR protocol with DNA extracted from two blood storage devices obtained from Giemsa-stained thick blood smears and filter-papers was used for malaria diagnosis. The extracted DNA was used as a template to amplify approximately 100 bp species-specific sequences of the small subunit of the ribosomal RNA (18S SSU rRNA) of Plasmodium sp. The prevalence of single and mixed infections was examined in a cross-sectional survey carried out among 369 miners living in the district of Apiacás, Mato Grosso State. The parasitemia levels detected by microscopic examination were compared to the PCR results.ResultsDNA samples isolated from blood on filter-paper allowed the detection and identification of Plasmodium in 165 (44.7%) of the 369 individuals evaluated, while only 62 (16.8%) had positive results using DNA obtained from thick smears, a similar rate observed by microscopic examination. The sensitivities of PCR using DNA from blood smears and filter-papers were 65% and 73.0%, respectively. Low parasite infections (below 20 parasites/µL blood) were not detected when thick blood smears were used as a DNA source.ConclusionsAlthough the blood preserved as thick blood smears provides an alternative and useful tool for malaria molecular diagnosis, its relatively poor performance at low level parasitemias impairs the correct determination of malaria prevalence in epidemiological studies. However, the results obtained in the present study confirm that the use of filter-paper to collect blood is useful for field studies.

Altered platelet indices as potential markers of severe and complicated malaria caused by Plasmodium vivax: a cross-sectional descriptive study

BackgroundThis study described altered platelet indices in patients with acute malaria caused by Plasmodium vivax and determined whether these alterations are associated with warning signs of severe and complicated malaria.MethodsA total of 186 patients attending the Malaria Clinic at the University Hospital from the Federal University of Mato Grosso, Brazil, between 2008 and 2013 were included in this study. After parasitological confirmation of exclusive infection by P. vivax, blood cell counts and platelet indices were determined. Disease gravity was evaluated on the basis of classic signs of Plasmodium falciparum severe malaria, including severe anemia, or by changes in serum levels of glucose, bilirubin, aminotransferases and creatinine at the time of the patient’s admission. Patients with a longer duration of symptoms or those identified as primo infected were considered potential candidates for evolution into the severe form of malaria.ResultsThe mean platelet volume (MPV), platelet distribution width (PDW), and plateletcrit (PCT) values exhibited significant variability. A significant inverse relationship was observed between parasitaemia and PCT. Patients with warning signs for evolution into severe disease, with primo infection, or presenting with symptoms for over three days had the highest MPV and PDW. The adjusted analyses showed the presence of warning signs for the development of severe and complicated malaria remained independently linked to elevated MPV and PDW.ConclusionAltered platelet indices should be analysed as potential markers for the severity of malaria caused by P. vivax. Future studies with appropriate methodology for prognostic evaluation could confirm the potential use of these indices in clinical practice.

Genetic variability and natural selection at the ligand domain of the Duffy binding protein in brazilian Plasmodium vivax populations

BackgroundPlasmodium vivax malaria is a major public health challenge in Latin America, Asia and Oceania, with 130-435 million clinical cases per year worldwide. Invasion of host blood cells by P. vivax mainly depends on a type I membrane protein called Duffy binding protein (PvDBP). The erythrocyte-binding motif of PvDBP is a 170 amino-acid stretch located in its cysteine-rich region II (PvDBPII), which is the most variable segment of the protein.MethodsTo test whether diversifying natural selection has shaped the nucleotide diversity of PvDBPII in Brazilian populations, this region was sequenced in 122 isolates from six different geographic areas. A Bayesian method was applied to test for the action of natural selection under a population genetic model that incorporates recombination. The analysis was integrated with a structural model of PvDBPII, and T- and B-cell epitopes were localized on the 3-D structure.ResultsThe results suggest that: (i) recombination plays an important role in determining the haplotype structure of PvDBPII, and (ii) PvDBPII appears to contain neutrally evolving codons as well as codons evolving under natural selection. Diversifying selection preferentially acts on sites identified as epitopes, particularly on amino acid residues 417, 419, and 424, which show strong linkage disequilibrium.ConclusionsThis study shows that some polymorphisms of PvDBPII are present near the erythrocyte-binding domain and might serve to elude antibodies that inhibit cell invasion. Therefore, these polymorphisms should be taken into account when designing vaccines aimed at eliciting antibodies to inhibit erythrocyte invasion.

Immunoglobulin GM 3 23 5,13,14 phenotype is strongly associated with IgG1 antibody responses to Plasmodium vivax vaccine candidate antigens PvMSP1-19 and PvAMA-1

BackgroundHumoral immune responses play a key role in the development of immunity to malaria, but the host genetic factors that contribute to the naturally occurring immune responses to malarial antigens are not completely understood. The aim of the present investigation was to determine whether, in subjects exposed to malaria, GM and KM allotypes--genetic markers of immunoglobulin γ and κ-type light chains, respectively--contribute to the magnitude of natural antibody responses to target antigens that are leading vaccine candidates for protection against Plasmodium vivax.MethodsSera from 210 adults, who had been exposed to malaria transmission in the Brazilian Amazon endemic area, were allotyped for several GM and KM determinants by a standard hemagglutination-inhibition method. IgG subclass antibodies to P. vivax apical membrane antigen 1 (PvAMA-1) and merozoite surface protein 1 (PvMSP1-19) were determined by an enzyme-linked immunosorbent assay. Multiple linear regression models and the non-parametric Mann-Whitney test were used for data analyses.ResultsIgG1 antibody levels to both PvMSP1-19 and PvAMA-1 antigens were significantly higher (P = 0.004, P = 0.002, respectively) in subjects with the GM 3 23 5,13,14 phenotype than in those who lacked this phenotype.ConclusionsResults presented here show that immunoglobulin GM allotypes contribute to the natural antibody responses to P. vivax malaria antigens. These findings have important implications for the effectiveness of vaccines containing PvAMA-1 or PvMSP1-19 antigens. They also shed light on the possible role of malaria as one of the evolutionary selective forces that may have contributed to the maintenance of the extensive polymorphism at the GM loci.

Improving N-terminal protein annotation of Plasmodium species based on signal peptide prediction of orthologous proteins

BackgroundSignal peptide is one of the most important motifs involved in protein trafficking and it ultimately influences protein function. Considering the expected functional conservation among orthologs it was hypothesized that divergence in signal peptides within orthologous groups is mainly due to N-terminal protein sequence misannotation. Thus, discrepancies in signal peptide prediction of orthologous proteins were used to identify misannotated proteins in five Plasmodium species.MethodsSignal peptide (SignalP) and orthology (OrthoMCL) were combined in an innovative strategy to identify orthologous groups showing discrepancies in signal peptide prediction among their protein members (Mixed groups). In a comparative analysis, multiple alignments for each of these groups and gene models were visually inspected in search of misannotated proteins and, whenever possible, alternative gene models were proposed. Thresholds for signal peptide prediction parameters were also modified to reduce their impact as a possible source of discrepancy among orthologs. Validation of new gene models was based on RT-PCR (few examples) or on experimental evidence already published (ApiLoc).ResultsThe rate of misannotated proteins was significantly higher in Mixed groups than in Positive or Negative groups, corroborating the proposed hypothesis. A total of 478 proteins were reannotated and change of signal peptide prediction from negative to positive was the most common. Reannotations triggered the conversion of almost 50% of all Mixed groups, which were further reduced by optimization of signal peptide prediction parameters.ConclusionsThe methodological novelty proposed here combining orthology and signal peptide prediction proved to be an effective strategy for the identification of proteins showing wrongly N-terminal annotated sequences, and it might have an important impact in the available data for genome-wide searching of potential vaccine and drug targets and proteins involved in host/parasite interactions, as demonstrated for five Plasmodium species.

Serum lipids in Brazilian children and adolescents: determining their reference intervals

BackgroundDemographic, geographic, environmental and genetic factors influence lipids. In many countries, the normal lipid ranges for laboratory tests are based on references from American children and adolescents. In this work, we determined the reference intervals (RIs) for total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), non-high-density lipoprotein cholesterol (nHDL-c), low-density lipoprotein cholesterol (LDL-c) and triglycerides (TG) in Brazilian healthy children and adolescents.MethodsA cross-sectional study was conducted of 1,866 randomly sampled healthy children and adolescents from kindergartens and schools. Blood samples were collected after a variable period of fasting based on the age of the participant. The upper cut-off points were the 75th and 95th percentiles for TC, nHDL-c, LDL-c and TG. The 10th percentile (low) was used as the bottom level for HDL-c. Non-parametric tests were used for statistical analyses.ResultsThe following RI and 75th and 95th percentiles were observed for each age interval. The 95th percentile values obtained for TC were: 1 to 2 years, 189 mg/dL, 3 to 8 years, 199 mg/dL; 9 to 12 years, 205 mg/dL. For the nHDL c, the only age group 1 to 12 years, this percentile value was 150 mg/dL. For the LDL-cholesterol, the values corresponding to the percentiles above, aged 1 to 8 years and 9 to 12 years, were 132 mg/dL 139 mg/dL, respectively. For the triglycerides, the values corresponding to 95th percentile were: 1 year, 189 mg/dL; 2 to 5 years, 139 mg/dL; 6 to 12 years, 139 mg/dL . The 10th percentiles for HDL-c were 24 mg/dL, 28 mg/dL, 32 mg/dL and 36 mg/dL for children 1, 2, 3 and 4-12 years old, respectively.ConclusionsThe lipid reference intervals defined in the studied Brazilian children and adolescents differ from those recommended by the international literature and should be used for clinical decisions contributing to improve the diagnosis in this particular group in our country.

Plasmodium vivax infection: atypical memory B cells are expanded and associated with the persistence of Duffy binding protein II (DBPII) antibody response

Antibody responses generated during malaria infection represent an important component of acquired clinical immunity. Despite that, B cell subpopulations induced by the Plasmodium vivax (Pv) infection remains largely unknown. Here, we demonstrated that activated as well as “atypical” memory B cells (MBCs) are expanded in peripheral blood of Pv-exposed individuals, but their frequencies were not associated with acute infection. Aiming to investigate the association between peripheral B cells subsets and Pv-specific antibodies, we further followed-up 34 individuals exposed to P. vivax in the Brazilian Amazon area, an area of markedly unstable malaria transmission; after three cross-sectional survey (at 6-months intervals), ELISA-detected specific IgG (AMA-1, MSP1-19, DBPII) allowed the classification of those individuals as non-responder (NR), temporary (TR) or persistent responder (PR). For AMA-1 and MSP1-19 serological groups, the frequencies of MBCs (classical and atypical) and plasma cells (PCs) were similar among the groups. For DBPII group, we found a trend toward decreases classical MBCs according to the antibody response (NR>TR>PR). On the other hand, the frequencies of atypical MBCs increased according to the presence and persistence of DBPII antibody response (PR>TR>NR). Altogether, these results showed that atypical MBCs are expanded in Pv-exposed individuals (infected and non-infected), and it seems to be associated with the persistence of DBPII antibody response. Although preliminary, these results suggest that atypical MBCs contribute in generation of malarial antibody responses and provide insight into the role of atypical MBCs in P. vivax malaria immunity.

Genetic variability of Plasmodium vivax from primary–relapses paired samples

Background Human malaria is caused by protozoa from the genus Plasmodium. Approximately 40% of world population is at risk of infection. Plasmodium vivax is the most worldwide distributed human Plasmodium species. In Brazil, more than 300,000 cases of the disease were recorded in 2010 and 86% were caused by Plasmodium vivax. P. vivax infections are characterized by hepatic dormant forms, the hypnozoites and those are activated in varying intervals of time resulting in the typical relapses. Little is known about the mechanism of latency and activation of hypnozoites associated to relapses. The scarcity of genetic markers for P. vivax has hampered the analysis of important parasite phenotypes, such as patterns of relapse. In this context, the aim of this work was to study the variability of the parasites of primary infections and relapses from the same patients. Materials and methods Primary-Relapse paired samples of 30 patients were genotyped using 10 molecular markers (8 microsatellites and blocks 2 and 10 of MSP-1) by capillary electrophoresis on an automated DNA sequencer. Moreover, the presence of multiple infections was confirmed by cloning of amplicons and genotyping of different colonies (mean of 10 colonies per PCR product). Results The majority of parasites showed distinct haplotypes in relapses compared to primary infection. It was demonstrated a high frequency of multiple-clone infections both in primary infection and relapse. A variation of predominant alleles among distinct markers in different malaria recidives of the same individual was observed. Therefore, haplotypes in relapse could also be identified in primary infections as a rare alleles. Conclusions Altogether our findings suggest that mechanisms involved in hypnozoites activation might not be based only on parasites genetic programming but also on host/enviroment factors.