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1.
Exp Parasitol ; 222: 108065, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33428893

RESUMO

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes.


Assuntos
Epitopos Imunodominantes/isolamento & purificação , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Proteômica/métodos , Sequência de Aminoácidos , Antígenos de Protozoários/isolamento & purificação , Western Blotting , Biologia Computacional/métodos , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Epitopos/isolamento & purificação , Humanos , Immunoblotting , Leishmaniose Visceral/imunologia , Conformação Molecular , Estrutura Secundária de Proteína , Proteômica/normas , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Testes Sorológicos/métodos , Testes Sorológicos/normas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
2.
PLoS One ; 15(12): e0243943, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33332459

RESUMO

Developing a vaccine against Plasmodium falciparum malaria has been challenging, primarily due to high levels of antigen polymorphism and a complex parasite lifecycle. Immunization with the P. falciparum merozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 has been shown to give rise to growth inhibitory and synergistic antisera. Therefore, these five merozoite proteins are considered to be promising candidates for a second-generation multivalent malaria vaccine. Nevertheless, little is known about IgG and IgM responses to these antigens in populations that are naturally exposed to P. falciparum. In this study, serum samples from clinically immune adults and malaria exposed children from Ghana were studied to compare levels of IgG and IgM specific for PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5. All five antigens were found to be specifically recognized by both IgM and IgG in serum from clinically immune adults and from children with malaria. Longitudinal analysis of the latter group showed an early, transient IgM response that was followed by IgG, which peaked 14 days after the initial diagnosis. IgG levels and parasitemia did not correlate, whereas parasitemia was weakly positively correlated with IgM levels. These findings show that IgG and IgM specific for merozoite antigens PfMSRP5, PfSERA9, PfRAMA, PfCyRPA and PfRH5 are high in children during P. falciparum malaria, but that the IgM induction and decline occurs earlier in infection than that of IgG.


Assuntos
Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Adolescente , Adulto , Idoso , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Vacinas Antimaláricas/imunologia , Malária Falciparum/parasitologia , Masculino , Merozoítos/imunologia , Merozoítos/patogenicidade , Pessoa de Meia-Idade , Plasmodium falciparum/patogenicidade , Adulto Jovem
3.
Artigo em Inglês | MEDLINE | ID: mdl-33375379

RESUMO

Histidine-rich proteins 2 and 3 gene (pfhrp2 and pfhrp3) deletions affect the efficacy of rapid diagnostic tests (RDTs) based on the histidine-rich protein 2 (HRP2), compromising the correct identification of the Plasmodium falciparum species. Therefore, molecular surveillance is necessary for the investigation of the actual prevalence of this phenomenon and the extent of the disappearance of these genes in these areas and other South American countries, thus guiding national malaria control programs on the appropriate use of RDTs. This study aimed to evaluate the pfhrp2 and pfhrp3 gene deletion in P. falciparum in endemic areas of the Brazilian Amazon. Aliquots of DNA from the biorepository of the Laboratory of Basic Research in Malaria, Evandro Chagas Institute, with a positive diagnosis for P. falciparum infection as determined by microscopy and molecular assays, were included. Monoinfection was confirmed by nested-polymerase chain reaction assay, and DNA quality was assessed by amplification of the merozoite surface protein-2 gene (msp2). The pfhrp2 and pfhrp3 genes were amplified using primers for the region between exons 1 and 2 and for all extension of exon 2. Aliquots of DNA from 192 P. falciparum isolates were included in the study, with 68.7% (132/192) from the municipality of Cruzeiro do Sul (Acre) and 31.3% (60/192) from Manaus (Amazonas). Of this total, 82.8% (159/192) of the samples were considered of good quality. In the state of Acre, 71.7% (71/99) showed pfhrp2 gene deletion and 94.9% (94/99) showed pfhrp3 gene deletion, while in the state of Amazonas, 100.0% (60/60) of the samples showed pfhrp2 gene deletion and 98.3% (59/60) showed pfhrp3 gene deletion. Moreover, 79.8% (127/159) of isolates displayed gene deletion. Our findings confirm the presence of a parasite population with high frequencies of pfhrp2 and pfhrp3 gene deletions in the Brazilian Amazon region. This suggests reconsidering the use of HRP2-based RDTs in the Acre and Amazonas states and calls attention to the importance of molecular surveillance and mapping of pfhrp2/pfhrp3 deletions in this area and in other locations in the Amazon region to guarantee appropriate patient care, control and ultimately contribute to achieving P. falciparum malaria elimination.


Assuntos
Plasmodium falciparum/genética , Proteínas/genética , Proteínas de Protozoários , Antígenos de Protozoários/genética , Brasil/epidemiologia , Testes Diagnósticos de Rotina , Deleção de Genes , Humanos , Malária Falciparum , Proteínas de Protozoários/genética
4.
PLoS Negl Trop Dis ; 14(12): e0008963, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33382690

RESUMO

Diagnosis of a first-time visceral leishmaniasis (VL) infection in Ethiopia is established by use of a rapid diagnostic test (RDT) detecting antibodies against rK39, direct agglutination test (DAT) and microscopy according to the national algorithm. The performance of individual tests and algorithm is variable and depends on several factors, one being HIV status. Limited data are available on the performance of tests in VL-HIV coinfected patients. Assessment of the performance of DAT (ITM-A), rK39 ELISA (Serion) and six RDT (Onsite Leishmania Ab CTK, Antigen ICT Xinjier, IT Leish Biorad, Kalazar Detect Inbios, rK39 IgG1 Coris, rk28 IgG1 Coris) for the diagnosis of VL was done on a panel of 91 stored serum and plasma samples of 'first-episode' suspected VL patients, with HIV coinfection (n = 51) and without (n = 40). A combined reference standard was used: either positive microscopy on tissue aspirates, or in case of negative microscopy, positive PCR results on the aspirate slide. Additionally, endemic healthy controls (n = 20), non-endemic controls (n = 10) and patients with confirmed malaria infection (n = 10) were tested for specificity evaluation. Sensitivities ranged from 69.2% for DAT (applied cut-off ≥ 1/3200) to 92.2% for the Onsite RDT, whereas specificities ranged from 20.0% for Kalazar Antigen ICT to 100% for IT Leish and rK39 IgG1. Sensitivities from all assays decreased upon stratification according to HIV status but was only significantly different for rK39 Serion ELISA (p-value 0.0084) and the Onsite RDT (p-value 0.0159). In conclusion, performance of commercially available assays for VL on samples from Northern-Ethiopian patients varied widely with a substantial decrease in sensitivity in the VL-HIV coinfected group. Clear guidelines on minimal performance criteria of individual tests and algorithms are needed, as well as which reference standard should be used to determine the performance.


Assuntos
Antígenos de Protozoários/imunologia , Infecções por HIV/complicações , Leishmania/imunologia , Leishmaniose Visceral/diagnóstico , Testes de Aglutinação , Testes Diagnósticos de Rotina , Ensaio de Imunoadsorção Enzimática , Etiópia/epidemiologia , Humanos , Leishmaniose Visceral/parasitologia , Padrões de Referência
5.
PLoS Pathog ; 16(8): e1008772, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866214

RESUMO

The tick-borne apicomplexan parasite, Babesia bovis, a highly persistent bovine pathogen, expresses VESA1 proteins on the infected erythrocyte surface to mediate cytoadhesion. The cytoadhesion ligand, VESA1, which protects the parasite from splenic passage, is itself protected from a host immune response by rapid antigenic variation. B. bovis relies upon segmental gene conversion (SGC) as a major mechanism to vary VESA1 structure. Gene conversion has been considered a form of homologous recombination (HR), a process for which Rad51 proteins are considered pivotal components. This could make BbRad51 a choice target for development of inhibitors that both interfere with parasite genome integrity and disrupt HR-dependent antigenic variation. Previously, we knocked out the Bbrad51 gene from the B. bovis haploid genome, resulting in a phenotype of sensitivity to methylmethane sulfonate (MMS) and apparent loss of HR-dependent integration of exogenous DNA. In a further characterization of BbRad51, we demonstrate here that ΔBbrad51 parasites are not more sensitive than wild-type to DNA damage induced by γ-irradiation, and repair their genome with similar kinetics. To assess the need for BbRad51 in SGC, RT-PCR was used to observe alterations to a highly variant region of ves1α transcripts over time. Mapping of these amplicons to the genome revealed a significant reduction of in situ transcriptional switching (isTS) among ves loci, but not cessation. By combining existing pipelines for analysis of the amplicons, we demonstrate that SGC continues unabated in ΔBbrad51 parasites, albeit at an overall reduced rate, and a reduction in SGC tract lengths was observed. By contrast, no differences were observed in the lengths of homologous sequences at which recombination occurred. These results indicate that, whereas BbRad51 is not essential to babesial antigenic variation, it influences epigenetic control of ves loci, and its absence significantly reduces successful variation. These results necessitate a reconsideration of the likely enzymatic mechanism(s) underlying SGC and suggest the existence of additional targets for development of small molecule inhibitors.


Assuntos
Antígenos de Protozoários , Babesia bovis , Conversão Gênica/imunologia , Genoma de Protozoário/imunologia , Proteínas de Protozoários , Rad51 Recombinase , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Babesia bovis/genética , Babesia bovis/imunologia , DNA de Protozoário/genética , DNA de Protozoário/imunologia , Haploidia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Rad51 Recombinase/genética , Rad51 Recombinase/imunologia
6.
PLoS One ; 15(9): e0238840, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32925980

RESUMO

Diagnosis of visceral leishmaniasis (VL) relies on invasive and risky aspirate procedures, and confirmation of cure after treatment is unreliable. Detection of Leishmania donovani antigens in urine has the potential to provide both a non-invasive diagnostic and a test of cure. We searched for L. donovani antigens in urine of VL patients from India and Sudan to contribute to the development of urine antigen capture immunoassays. VL urine samples were incubated with immobilised anti-L. donovani polyclonal antibodies and captured material was eluted. Sudanese eluted material and concentrated VL urine were analysed by western blot. Immunocaptured and immunoreactive material from Indian and Sudanese urine was submitted to mass spectrometry for protein identification. We identified six L. donovani proteins from VL urine. Named proteins were 40S ribosomal protein S9, kinases, and others were hypothetical. Thirty-three epitope regions were predicted with high specificity in the 6 proteins. Of these, 20 were highly specific to Leishmania spp. and are highly suitable for raising antibodies for the subsequent development of an antigen capture assay. We present all the identified proteins and analysed epitope regions in full so that they may contribute to the development of non-invasive immunoassays for this deadly disease.


Assuntos
Anticorpos Antiprotozoários/urina , Antígenos de Protozoários/urina , Leishmania donovani/imunologia , Leishmaniose Visceral/diagnóstico , Proteínas de Protozoários/urina , Adulto , Anticorpos Antiprotozoários/imunologia , Anticorpos Antiprotozoários/isolamento & purificação , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Estudos de Casos e Controles , Humanos , Índia/epidemiologia , Leishmania donovani/isolamento & purificação , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/urina , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação
7.
PLoS One ; 15(9): e0238749, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32886699

RESUMO

INTRODUCTION: False-negative malaria rapid diagnostic test (RDT) results amongst symptomatic malaria patients are detrimental as they could lead to ineffective malaria case management. This study determined the nationwide contribution of parasites with Pfhrp2 and Pfhrp 3 gene deletions to false negative malaria RDT results in Ghana. METHODS: This was a cross sectional study where whole blood (~2 ml) was collected from patients presenting with malaria symptoms at 100 health facilities in all the regions in Ghana from May to August 2018. An aliquot of the blood was used to prepare thin and thick blood smears, filter paper blood spots (DBS) and spot a PfHRP 2 RDT kit. The remaining blood was separated into plasma and blood cells and stored at -20°C. Plasmodium parasite density and species identity was estimated from the blood smears. Plasmodium falciparum specific 18S rRNA PCR, merozoite surface protein (msp 1) and glutamate rich protein (glurp) gene PCR were used to identify P. falciparum positive samples, which were subjected to Pfhrp 2/3 exon1-2 and exon2 genotyping. RESULTS: Of the 2,860 microscopically P. falciparum positive patients analyzed, 134 (4.69%) had false negative P. falciparum specific RDT results. Samples for PCR analysis was available for 127 of the false negative patients, and the analysis identified 116 (91.3%) as positive for P. falciparum. Only 58.1% (79/116) of the false negative RDT samples tested positive by msp 1 and glurp PCR. Genotyping of exon 1-2 and exon 2 of the Pfhrp 2 gene identified 12.9% (10/79) and 39.5% (31/79) of samples respectively to have deletions. Genotyping exon 1-2 and exon 2 of the Pfhrp 3 gene identified 15.2% (12/79) and 40.5% (32/79) of samples respectively to have deletions. Only 5% (4/79) of the false negative samples had deletions in both exon 1-2 and exon 2 of the Pfhrp 2 gene. Out of the 49 samples that tested positive for aldolase by luminex, 32.6% (16/49) and) had deletions in Pfhrp 2 exon 2 and 2% (1/49) had deletions in both exon 2 and exon 1-2 of the Pfhrp 2 gene. CONCLUSIONS: The low prevalence of false negative RDT test results provides assurance that PfHRP 2 based malaria RDT kits remain effective in diagnosing symptomatic malaria patients across all the Regions of Ghana. Although there was a low prevalence of parasites with deletions in exon 2 and exon 1-2 of the Pfhrp 2 gene the prevalence of parasites with deletions in Pfhrp 2 exon 2 was about a third of the false negative RDT results. The need to ensure rapid, accurate and reliable malaria diagnosis requires continuous surveillance of parasites with Pfhrp 2 gene deletions.


Assuntos
Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Deleção de Genes , Malária Falciparum/diagnóstico , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Adulto , Antígenos de Protozoários/imunologia , Reações Falso-Negativas , Feminino , Técnicas de Genotipagem , Gana/epidemiologia , Humanos , Malária Falciparum/epidemiologia , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/imunologia , Plasmodium falciparum/fisiologia , Adulto Jovem
8.
Parasitol Res ; 119(11): 3639-3648, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32930858

RESUMO

Due to its wide presence in apicomplexan parasites as well as high polymorphism and antigenic diversity, the variable merozoite surface antigen (VMSA) family in Babesia sp. has attracted increasing attention of researchers. Here, all the reported VMSA genes of Babesia spp. were obtained from GenBank, and multiple alignments were performed by using conserved regions to blast the Babesia orientalis genome database (unpublished data). Five MSA genes (named MSA-2a1, MSA-2a2, MSA-2c1, MSA-1, and MSA-2c2, respectively) were identified, sequenced, and cloned from B. orientalis, which were shown to encode proteins with open reading frames ranging in size from 266 (MSA-2c1) to 317 (MSA-1) amino acids. All the five proteins contain an MSA-2c superfamily conserved domain, with an identical signal peptide and glycosyl phosphatidyl inositol (GPI)-anchor for each of them. The five proteins were also predicted to contain B cell epitopes, with only three for BoMSA-2c1, the smallest protein in the BoVMSA family, while at least six for each of the others. Notably, BoMSA-2a1 has 2 identical copies, a specific phenomenon only present in B. orientalis. This research has determined the MSA genes of B. orientalis and provides a genetic basis for further research of functional genes in B. orientalis.


Assuntos
Antígenos de Protozoários/genética , Babesia/genética , Proteínas de Protozoários/genética , Animais , Antígenos de Protozoários/imunologia , Antígenos de Superfície/genética , Babesia/imunologia , Epitopos de Linfócito B , Glicosilfosfatidilinositóis/análise , Proteína 1 de Superfície de Merozoito/genética , Merozoítos/química , Merozoítos/imunologia , Fases de Leitura Aberta , Polimorfismo Genético , Proteínas de Protozoários/imunologia
9.
J Vis Exp ; (162)2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32831311

RESUMO

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.


Assuntos
Anticorpos Antiprotozoários/análise , Bioensaio/métodos , Citometria de Fluxo/métodos , Parasitos/imunologia , Fagocitose , Plasmodium falciparum/imunologia , Animais , Antígenos de Protozoários/imunologia , Eritrócitos/parasitologia , Feminino , Humanos , Malária Falciparum/sangue , Malária Falciparum/parasitologia , Proteínas Opsonizantes/metabolismo , Gravidez , Receptores Fc/metabolismo , Reprodutibilidade dos Testes , Células THP-1
10.
Am J Trop Med Hyg ; 103(5): 1902-1909, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32840197

RESUMO

In malaria-endemic countries, rapid diagnostic tests (RDTs) targeting Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and lactate dehydrogenase (PfLDH) have been widely used. However, little is known regarding the diagnostic performances of these RDTs in the Assosa zone of northwest Ethiopia. The objective of this study was to determine the diagnostic performances of PfHRP2 and PfLDH RDTs using microscopy and quantitative PCR (qPCR) as a reference test. A health facility-based cross-sectional study design was conducted from malaria-suspected study participants at selected health centers from November to December 2018. Finger-prick blood samples were collected for microscopy, RDTs, and qPCR method. The prevalence of P. falciparum was 26.4%, 30.3%, and 24.1% as determined by microscopy, PfHRP2 RDT, and PfLDH RDT, respectively. Compared with microscopy, the sensitivity and specificity of the PfHRP2 RDT were 96% and 93%, respectively, and those of the PfLDH RDT were 89% and 99%, respectively. Compared with qPCR, the specificity of the PfHRP2 RDT (93%) and PfLDH RDT (98%) was high, but the sensitivity of the PfHRP2 RDT (77%) and PfLDH RDT (70%) was relatively low. These malaria RDTs and reference microscopy methods showed reasonable agreement with a kappa value above 0.85 and provided accurate diagnosis of P. falciparum malaria. Thus, the current malaria RDT in the Ministry of Health program can be used in the Assosa zone of Ethiopia. However, continuous monitoring of the performance of PfHRP2 RDT is important to support control and elimination of malaria in Ethiopia.


Assuntos
Antígenos de Protozoários/imunologia , Testes Diagnósticos de Rotina/métodos , L-Lactato Desidrogenase/imunologia , Malária Falciparum/diagnóstico , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Antígenos de Protozoários/genética , Criança , Pré-Escolar , Estudos Transversais , Demografia , Etiópia , Feminino , Geografia , Humanos , L-Lactato Desidrogenase/genética , Malária Falciparum/parasitologia , Masculino , Microscopia , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , Sensibilidade e Especificidade , Adulto Jovem
11.
PLoS One ; 15(8): e0237671, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32797068

RESUMO

In high malaria transmission settings, the use of sulfadoxine-pyrimethamine-based intermittent preventive treatment during pregnancy (IPTp-SP) has resulted in decreased antibody (Ab) levels to VAR2CSA. However, information of Ab levels in areas of low or intermediate malaria transmission after long-term implementation of IPTp-SP is still lacking. The present study sought to evaluate antibody prevalence and levels in women at delivery in Etoudi, a peri-urban area in the capital of Yaoundé, Cameroon, that is a relatively low-malaria transmission area. Peripheral plasma samples from 130 pregnant women were collected at delivery and tested for IgG to the full-length recombinant VAR2CSA (FV2) and its most immunogenic subdomain, DBL5. The study was conducted between 2013 and 2015, approximately ten years after implementation of IPTp-SP in Cameroon. About 8.6% of the women attending the clinic had placental malaria (PM). One, two or 3 doses of SP did not impact significantly on either the percentage of women with Ab to FV2 and DBL5 or Ab levels in Ab-positive women compared to women not taking SP. The prevalence of Ab to FV2 and DBL5 was only 36.9% and 36.1%, respectively. Surprisingly, among women who had PM at delivery, only 61.5% and 57.7% had Ab to FV2 and DBL5, respectively, with only 52.9% and 47.1% in PM-positive paucigravidae and 77.7% of multigravidae having Ab to both antigens. These results suggest that long-term implementation of IPTp-SP in a low-malaria transmission area results in few women having Ab to VAR2CSA.


Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Antimaláricos/uso terapêutico , Malária Falciparum/prevenção & controle , Complicações Parasitárias na Gravidez/prevenção & controle , Pirimetamina/uso terapêutico , Sulfadoxina/uso terapêutico , Adulto , Anticorpos Antiprotozoários/sangue , Camarões/epidemiologia , Combinação de Medicamentos , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Malária/sangue , Malária/epidemiologia , Malária/imunologia , Malária/prevenção & controle , Malária Falciparum/sangue , Malária Falciparum/epidemiologia , Malária Falciparum/imunologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/imunologia , Gravidez , Complicações Parasitárias na Gravidez/sangue , Complicações Parasitárias na Gravidez/epidemiologia , Complicações Parasitárias na Gravidez/imunologia , Adulto Jovem
12.
BMC Infect Dis ; 20(1): 573, 2020 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-32758164

RESUMO

BACKGROUND: Malaria during pregnancy leads to serious adverse effects on mothers and the fetus. Approximately 25 million pregnant women in sub-Saharan Africa live at risk of malaria. This study would help to achieve Sustainable Development Goals (SDGs) by improving programs that deal with the prevention of malaria. Therefore, this study aimed to assess the prevalence and associated factors of malaria among pregnant women. METHODS: A community-based cross-sectional study was conducted from July to August 2018 in Sherkole district, West Ethiopia. A multi-stage sampling technique was used to select 504 pregnant women. The interviewer-administered semi-structured questionnaire was used for data collection. Malaria was also diagnosed using a rapid diagnostic test. The data was entered using EPI info version 7.2.2.2 and transferred to SPSS version 20 for analysis. Descriptive statistics were done using frequency and percentages. Both bivariable and multivariable logistic regression models were employed. Variables having p-value < 0.2 were included in the final multivariable model. Variables having p-values < 0.05 from the multivariable model were considered to be significantly associated with the dependent variable. The adjusted odds ratio with its 95% confidence interval (CI) was used as a measure of association. RESULTS: Of the total 498 pregnant women who participated in this study, 51(10.2, 95% CI: 7.72-13.24) were found to have malaria. Of these, 46 (90.2%) and 5 (9.8%) were caused by Plasmodium falciparum and Plasmodium vivax, respectively. Decreasing Age (Adjusted Odds Ratio (AOR) 0.78; 95% CI 0.67-0.911), not using insecticide-treated bed net (ITN) (AOR 12.5; 95% CI 4.86-32.21), lack of consultation and health education about malaria prevention (AOR 7.18; 95% CI 2.74-18.81), being on second-trimester pregnancy (AOR 7.58; 95% CI 2.84-20.2), gravidae II (AOR 5.99; 95% CI 1.68-21.44) were found to be significantly associated with malaria during pregnancy. CONCLUSION: Malaria is still a public health problem among pregnant women in the Sherkole district. Age, ITN use, gravidity, gestational age, and health education had a significant association with malaria. Screening pregnant women for asymptomatic malaria infection and educating and consulting on the appropriate malaria preventive methods shall be provided.


Assuntos
Malária Falciparum/epidemiologia , Malária Vivax/epidemiologia , Plasmodium falciparum/imunologia , Plasmodium vivax/imunologia , Complicações Parasitárias na Gravidez/epidemiologia , Adolescente , Adulto , Antígenos de Protozoários/sangue , Infecções Assintomáticas , Estudos Transversais , Testes Diagnósticos de Rotina/métodos , Etiópia/epidemiologia , Feminino , Seguimentos , Humanos , Mosquiteiros Tratados com Inseticida , Modelos Logísticos , Malária Falciparum/prevenção & controle , Malária Vivax/prevenção & controle , Razão de Chances , Gravidez , Complicações Parasitárias na Gravidez/prevenção & controle , Prevalência , Fatores de Risco , Inquéritos e Questionários , Adulto Jovem
13.
PLoS Negl Trop Dis ; 14(7): e0008488, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716931

RESUMO

BACKGROUND: Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both on in silico gene design and experimental data. Starting from the previous identification of Leishmania infantum recombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy controls. A single non-specific reaction was seen with serum from individuals with tegumentary leishmaniasis. CONCLUSION: The newly described chimeric protein is potentially useful for the detection of both humans and dogs afflicted with VL, with its use in rapid tests necessary for validation as a new diagnostic tool.


Assuntos
Doenças do Cão/diagnóstico , Leishmaniose Visceral/veterinária , Testes Sorológicos/veterinária , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Doenças do Cão/sangue , Cães , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Humanos , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Proteínas de Protozoários/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Transcriptoma
14.
Mol Immunol ; 125: 104-114, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32659595

RESUMO

Leishmania are obligate protozoan parasites responsible for substantial public health problems in tropical and subtropical regions around the world, with L. braziliensis being one of the causative agents of American Tegumentary Leishmaniasis. Macrophages, fundamental cells in the innate inflammatory response against Leishmania, constitute a heterogeneous group with multiple activation phenotypes and functions. The outcome of this infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. The importance of lipids, the major components of cell membranes, goes beyond their basic structural functions. Lipid bioactive molecules have been described in Leishmania spp., and in the recent years the knowledge about the biological relevance of lipids in particular and their relationship with the immune response is expanding. The present work analyzes the biological effects of L. braziliensis lipids from lysed promastigotes (PRO) to mimic rapid modulatory processes that could occur in the initial steps of infection or the effects of lipids from lysed and incubated promastigotes (PROinc), simulating the parasite lipid degradation processes triggered after parasite lysis that might occur in the mammalian host. To perform these studies, lipid profiles of PRO and PROinc were compared with lipids from amastigotes under similar conditions (AMA and AMAinc), and the effect of these lipid extracts were analyzed on the induction of an inflammatory response in murine peritoneal macrophages: LB induction, COX-2, iNOS and Arginase expression, TNF-α, IL-10 and NO production, Arginase activity and M1/M2 markers mRNA induction.


Assuntos
Antígenos de Protozoários/imunologia , Leishmania braziliensis/imunologia , Leishmaniose/imunologia , Lipídeos/imunologia , Ativação de Macrófagos/imunologia , Animais , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C
15.
Mem Inst Oswaldo Cruz ; 115: e200067, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32667458

RESUMO

BACKGROUND Unfortunately, no any vaccine against leishmaniasis has been developed for human use. Therefore, a vaccine based on total Leishmania antigens could be a good and economic approach; and there are different methodologies to obtain these antigens. However, it is unknown whether the method to obtain the antigens affects the integrity and immune response caused by them. OBJECTIVES to compare the protein profile and immune response generated by total L. amazonensis antigens (TLA) produced by different methods, as well as to analyse the immune response and protection by a first-generation vaccine formulated with sonicated TLA (sTLA) and polyinosinic:polycytidylic acid [Poly (I:C)]. METHODS TLA were obtained by four different methodologies and their integrity and immune response were evaluated. Finally, sTLA was formulated with Poly (I:C) and their protective immune response was measured. FINDINGS sTLA presented a conserved protein profile and induced a strong immune response. In addition, Poly (I:C) improved the immune response generated by sTLA. Finally, sTLA + Poly (I:C) formulation provided partial protection against L. amazonensis infection. MAIN CONCLUSIONS The protein profile and immune response depend on the methodology used to obtain the antigens. Also, the formulation sTLA + Poly (I:C) provides partial protection against cutaneous leishmaniasis in mice.


Assuntos
Leishmania , Vacinas contra Leishmaniose , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/prevenção & controle , Vacinas Protozoárias/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Antígenos de Protozoários/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C
16.
Am J Trop Med Hyg ; 103(2): 778-784, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32602431

RESUMO

The ultrasensitive Alere Plasmodium falciparum Malaria Ag histidine-rich protein 2 rapid diagnostic test (Alere uRDT, Suwon City, South Korea) is a new diagnostic tool which is more expensive than other malaria rapid diagnostic tests (RDTs) routinely used in Ugandan clinics. The manufacturer recommends testing samples within 2 days and scoring results after 20 minutes, which may be impractical in high-volume resource-poor clinics. We compared testing by the Alere Ag rapid diagnostic test (uRDT), CareStart RDT, microscopy, and an ultrasensitive I8S rRNA quantitative reverse transcription polymerase chain reaction (qRT-PCR) using survey and clinical samples. For the Alere uRDT, we used survey blood samples stored at 4°C for 44 days and for some clinical samples deliberately scored results beyond 20 minutes. The Alere uRDT and qRT-PCR identified asymptomatic parasitemia cases in 56% and 72%, respectively, of survey samples originally scored as negative by the CareStart RDT. Using qRT-PCR as a gold standard, the Alere uRDT was superior to the CareStart RDT in estimating asymptomatic parasite prevalence in a cross-sectional survey (P = 0.007) and in detection of clinically significant malaria; both RDTs were comparable in detecting asymptomatic parasitemia in the clinic (P = 0.599). Scoring Alere uRDT results at 20 minutes produced valid results confirmed by the CareStart RDT, but there was a consistent background; scoring the Alere uRDT beyond 20 minutes produced false-positive results. The Alere uRDT outperformed the CareStart RDT (ACCESSBIO, Somerset, NJ) in a field survey in estimating malaria prevalence and in the clinic for symptomatic malarial illness. It produced reliable results using samples stored at 4°C for 44 days, but test results read beyond 20 minutes were invalid.


Assuntos
Antígenos de Protozoários/imunologia , Malária Falciparum/diagnóstico , Parasitemia/diagnóstico , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Infecções Assintomáticas , Criança , Pré-Escolar , Reações Falso-Positivas , Feminino , Humanos , Lactente , Masculino , Microscopia , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , RNA Ribossômico 18S/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Fatores de Tempo , Uganda
17.
Exp Parasitol ; 216: 107941, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32622940

RESUMO

Distinct antigens have been evaluated with diagnostic purpose for canine and human visceral leishmaniasis (VL), and variable sensitivity and specificity values have been obtained in the assays. In the present study, a Leishmania infantum hypothetical protein called LiHyG, which was identified in an immunoproteomics study in Leishmania infantum amastigote extracts by antibodies in VL dogs sera; was cloned, expressed, purified and evaluated as a recombinant protein (rLiHyG) for the diagnosis of canine and human disease. The recombinant amastigote-specific A2 protein (rA2) and a soluble L. infantum protein extract (SLA) were used as controls. For canine VL, the sensitivity values were of 100%, 57.29% and 48.57%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 81.43% and 88.57%, respectively. In addition, AUC values were of 1.00, 0.72 and 0.65, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 72.38% and 75.24%, respectively. For human VL, the sensitivity values were of 100%, 84.00% and 88.00%, when rLiHyG, rA2 and SLA were used, respectively, while the specificity values were of 100%, 58.75% and 73.75%, respectively. In addition, AUC values were of 1.00, 0.76 and 0.83, when rLiHyG, rA2 and SLA were used, respectively, while accuracy was of 100%, 64.8% and 66.6%, respectively. The prognostic role of rLiHyG in the human VL was also evaluated, by means of post-therapeutic serological follow-up with sera samples collected before and six months after treatment. Results showed that treated patients presented significant reductions in the anti-rLiHyG IgG, IgG1, and IgG2 antibody levels, with results being similar to those found in healthy subjects. Testing the rA2 protein and SLA as antigens, lower IgG, IgG1, and IgG2 levels were also found, although they were higher after treatment than those obtained for rLiHyG. In conclusion, results suggested that rLiHyG could be considered for future studies as a diagnostic and/or prognostic marker for canine and human VL.


Assuntos
Antígenos de Protozoários/isolamento & purificação , Doenças do Cão/parasitologia , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Adulto , Idoso , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Medula Óssea/parasitologia , Biologia Computacional , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Doenças do Cão/diagnóstico , Cães , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/química , Feminino , Humanos , Imunoglobulina G/sangue , Leishmania infantum/genética , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/veterinária , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas de Protozoários/química , Sensibilidade e Especificidade , Alinhamento de Sequência , Testes Sorológicos , Baço/parasitologia , Adulto Jovem
18.
Parasitol Res ; 119(9): 2907-2916, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32686022

RESUMO

The protozoan parasite Toxoplasma gondii secretes a number of dense granule proteins (GRAs) from the dense granule organelle to manipulate the host cell. Two of these effector proteins (GRA17 and GRA23) are involved in the trafficking of molecules between the parasitophorous vacuole (PV) and the host cell cytoplasm. However, their roles in establishing chronic infection remain obscured. In this study, CRISPR-Cas9 was used to delete gra17 or gra23 gene in T. gondii Pru strain (type II). The growth, the virulence, the ability to establish chronic infection, and the immunogenicity of the constructed mutant strains were investigated in Kunming mice. Pru:Δgra17 and Pru:Δgra23 mutants developed PVs with abnormal morphology and exhibited reduced growth rate, compared with the wild-type Pru strain. Deletion of gra17 abrogated acute infection and blocked cyst formation. Although the deletion of gra23 caused slight attenuation of the parasite virulence in mice, it caused a significant reduction in cyst formation. Immunization with Pru:Δgra17 induced high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (interleukin-2 [IL-2], IL-10, IL-12, and interferon gamma [IFN-γ]), which conferred significant protection in mice challenged with virulent type I (RH), ToxoDB#9 (PYS) strains, or less virulent type II (Pru) strain of T. gondii. These findings show that GRA17 and GRA23 play important roles in T. gondii chronic infection and that irreversible deletion of gra17 in T. gondii type II Pru strain can be a viable option for stimulating protective immunity to T. gondii infection.


Assuntos
Antígenos de Protozoários/imunologia , Citocinas/metabolismo , Proteínas de Protozoários/genética , Toxoplasma , Fatores de Virulência/genética , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/imunologia , Toxoplasma/patogenicidade , Toxoplasmose Animal/parasitologia , Virulência/genética
19.
PLoS Negl Trop Dis ; 14(7): e0008471, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32639964

RESUMO

In Brazil, Plasmodium vivax infection accounts for around 80% of malaria cases. This infection has a substantial impact on the productivity of the local population as the course of the disease is usually prolonged and the development of acquired immunity in endemic areas takes several years. The recent emergence of drug-resistant strains has intensified research on alternative control methods such as vaccines. There is currently no effective available vaccine against malaria; however, numerous candidates have been studied in the past several years. One of the leading candidates is apical membrane antigen 1 (AMA1). This protein is involved in the invasion of Apicomplexa parasites into host cells, participating in the formation of a moving junction. Understanding how the genetic diversity of an antigen influences the immune response is highly important for vaccine development. In this study, we analyzed the diversity of AMA1 from Brazilian P. vivax isolates and 19 haplotypes of P. vivax were found. Among those sequences, 33 nonsynonymous PvAMA1 amino acid sites were identified, whereas 20 of these sites were determined to be located in predicted B-cell epitopes. Nonsynonymous mutations were evaluated for their influence on the immune recognition of these antigens. Two distinct haplotypes, 5 and 16, were expressed and evaluated for reactivity in individuals from northern Brazil. Both PvAMA1 variants were reactive. Moreover, the IgG antibody response to these two PvAMA1 variants was analyzed in an exposed but noninfected population from a P. vivax endemic area. Interestingly, over 40% of this population had antibodies recognizing both variants. These results have implications for the design of a vaccine based on a polymorphic antigen.


Assuntos
Antígenos de Protozoários/genética , Malária Vivax/imunologia , Malária Vivax/parasitologia , Proteínas de Membrana/genética , Plasmodium vivax/genética , Proteínas de Protozoários/genética , Dicroísmo Circular , DNA de Protozoário/genética , Epitopos de Linfócito B , Haplótipos , Humanos , Malária Vivax/epidemiologia , Mutação , Plasmodium vivax/imunologia , Conformação Proteica , Proteínas Recombinantes
20.
PLoS One ; 15(7): e0236369, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702040

RESUMO

Histidine-rich protein 2 (HRP2) detecting rapid diagnostic tests (RDTs) have played an important role in enabling prompt malaria diagnosis in remote locations. However, emergence of pfhrp2 deleted parasites is threatening the efficacy of RDTs, and the World Health Organization (WHO) has highlighted surveillance of these deletions as a priority. Nested PCR is used to confirm pfhrp2 deletion but is costly and laborious. Due to spurious amplification of paralogue pfhrp3, the identity of nested exon 1 PCR product must be confirmed by sequencing. Here we describe a new one-step PCR method for detection of pfhrp2. To determine sensitivity and specificity, all PCRs were performed in triplicate. Using photo-induced electron transfer (PET) PCR detecting 18srRNA as true positive, one-step had comparable sensitivity of 95.0% (88.7-98.4%) to nested exon 1, 99.0% (94.6-99.9%) and nested exon 2, 98.0% (93.0-99.8%), and comparable specificity 93.8% (69.8-99.8%) to nested exon 1 100.0% (79.4-100.0%) and nested exon 2, 100.0% (74.4-100.0%). Sequencing revealed that one step PCR does not amplify pfhrp3. Logistic regression models applied to measure the 95% level of detection of the one-step PCR in clinical isolates provided estimates of 133p/µL (95% confidence interval (CI): 3-793p/µL) for whole blood (WB) samples and 385p/µL (95% CI: 31-2133 p/µL) for dried blood spots (DBSs). When considering protocol attributes, the one-step PCR is less expensive, faster and more suitable for high throughput. In summary, we have developed a more accurate PCR method that may be ideal for the application of the WHO protocol for investigating pfhrp2 deletions in symptomatic individuals presenting to health care facilities.


Assuntos
Antígenos de Protozoários/genética , Malária Falciparum/diagnóstico , Plasmodium falciparum/genética , Reação em Cadeia da Polimerase/métodos , Proteínas de Protozoários/genética , Testes Diagnósticos de Rotina , Deleção de Genes , Humanos , Malária Falciparum/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/patogenicidade
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