RESUMO
The accurate and efficient measurement of white blood cell (WBC) counts is vital for monitoring general patient health and can aid in the diagnosis of a range of possible infections or diseases. Even with their importance universally acknowledged, access to WBC counts is largely limited to those with access to phlebotomists and centralized clinical laboratories, which house the instruments that perform the tests. As a result, large populations of people (e.g., those that are home-bound or live in remote locations) lack facile access to testing. Dried blood spot (DBS) cards are often used to bridge these gaps in access to testing by offering the ability to collect blood at home for ambient shipping to laboratories. However, it is well understood that these cards, which are prepared from cellulose cardstocks without further modification, suffer from variabilities in accuracy and precision due to uncontrolled sample spreading and hematocrit effects, which have hindered their use to determine WBC counts. In this paper, we present a method to obtain an accurate WBC count using a patterned dried blood spot (pDBS) card, which comprises collection zones that meter volumes of dried blood. Using an input volume of 75 µL of whole blood, we demonstrate that, unlike the gold standard DBS card (Whatman 903), our pDBS design allows for the collection of replicate zones containing a reproducible, average volume of dried blood (12.1 µL, 7.8% CV) over the range of hematocrits from 25 to 55%. We then used qPCR to quantify the 18S rRNA gene to determine WBC counts from the volumes of blood that are metered in pDBS zones. We observe that WBC counts generated from our method are comparable to those measured with a HemoCue point-of-care WBC analyzer. Our approach to using pDBS cards as a blood collection device has the potential to support at-home sampling and other patient populations that need WBC counts but lack access to clinical facilities.
Assuntos
Coleta de Amostras Sanguíneas , Teste em Amostras de Sangue Seco , Humanos , Hematócrito , Teste em Amostras de Sangue Seco/métodos , Contagem de Leucócitos , CeluloseRESUMO
Sexual differentiation of malaria parasites from the asexual blood stage into gametocytes is an essential part of the life cycle, as gametocytes are the form that is taken up by the mosquito host. Because of the essentiality of this process for transmission to the mosquito, gametocytogenesis is an extremely attractive target for therapeutic interventions. The subject of this review is the considerable progress that has been made in recent years in elucidating the molecular mechanisms governing this important differentiation process. In particular, a number of critical transcription factors and epigenetic regulators have emerged as crucial elements in the regulation of commitment. The identification of these factors has allowed us to understand better than ever before the events occurring prior to and during commitment to sexual development and offers potential for new therapeutic interventions.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica , Plasmodium/crescimento & desenvolvimento , Plasmodium/genética , Redes Reguladoras de Genes , Fatores de Transcrição/metabolismoRESUMO
Antibody sequence repertoire analysis of plasma cells (PC) isolated before and 1 week after a vaccine provides time-specific snapshots of the antibody response. Comparison of the immunoglobulin (Ig) sequences pre- and post-vaccination allows analysis of maturation over time and identification of antigen specific Ig. Here we compare the Ig heavy chain (Ig-H) repertoire of circulating PCs isolated from 109 peripheral blood mononuclear cells (PBMC) collected by apheresis 1 week after a tetanus toxoid vaccine booster with the Ig-H repertoire of PCs collected 2 and 11 weeks prior to the booster. A total of 21,060 unique Ig nucleotide sequences encoding 14,307 unique heavy chain complementarity determining region 3 (CDR-H3) amino acid sequences, also called clonotypes, were identified. Only 466 clonotypes (3.3%) were present at all 3 time points. In contrast, 90% of the 30 highest frequency CDR-H3 regions at +1w were also identified at another time point and 50% were present at all time points, suggesting the rapid expansion of a memory B cell population. The tetanus toxoid specificity of the CDR-H3 region with the 7th highest frequency at +1w was confirmed using immunoprecipitation and mass spectroscopy, and two public tetanus toxoid-specific CDR-H3 regions were also overrepresented at +1w. In summary, we have used the tetanus vaccine model system to demonstrate that bulk PC Ig repertoire analysis can identify PC populations that expand and mature following antigen exposure. The application of this approach before and after clinical infections should advance our understanding of clinical protection and facilitate vaccine design.
Assuntos
Regiões Determinantes de Complementaridade/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Leucócitos Mononucleares/imunologia , Plasmócitos/imunologia , Toxoide Tetânico/imunologia , Vacinação/métodos , Regiões Determinantes de Complementaridade/imunologia , Voluntários Saudáveis , Humanos , Leucócitos Mononucleares/metabolismo , Toxoide Tetânico/administração & dosagemRESUMO
BACKGROUND: Red blood cell (RBC) polymorphisms are suggested to influence the course of Plasmodium falciparum malaria. Whereas some variants have been found to be protective, others have been found to enhance parasite development. This study evaluated the effect of variant haemoglobin (Hb) and ABO blood groups on P. falciparum merozoite invasion, multiplication rates as well as gametocyte development. METHODS: Approximately 2.5 mL of venous blood was collected from each participant. Flow cytometry was used to determine the in vitro merozoite invasion rates of NF54 parasites into the blood of 66 non-parasitaemic individuals with variant Hb genotypes (HbSS, HbSC) and blood groups (A, B, O), which were then compared with invasion into HbAA blood. The ex vivo asexual parasite multiplication and gametocyte production rates of parasites from 79 uncomplicated malaria patients with varying Hb genotypes (HbAS, HbAC and HbAA) were also estimated using microscopy. RESULTS: Merozoite invasion rates were significantly reduced by about 50% in RBCs containing HbSS and HbSC relative to HbAA cells. The presence of blood group O and B reduced the invasion rates of HbSS by about 50% and 60%, respectively, relative to HbSC but the presence of blood group A removed the inhibitory effect of HbSS. The initial parasite densities in uncomplicated malaria patients with Hb genotypes HbAS and HbAC cells were similar but significantly lower than those with genotype HbAA. The ex vivo parasite multiplication rate, gametocytaemia and gametocyte conversion rates followed a similar trend but did not reach statistical significance (p > 0.05). CONCLUSIONS: Parasite invasion rate into erythrocytes is dependent on both erythrocyte blood group antigen and haemoglobin genotype as blood group O and B provided protection via reduced merozoite invasion in RBCs containing HbSS relative to HbSC. Regardless of haemoglobin type, greater than 70% malaria patients had circulating ring stage parasites that differentiated into stage II gametocytes in 4 days.
Assuntos
Eritrócitos/parasitologia , Malária Falciparum/sangue , Plasmodium falciparum/crescimento & desenvolvimento , Adolescente , Adulto , Antígenos de Grupos Sanguíneos/classificação , Criança , Estudos Transversais , Feminino , Gana , Hemoglobinas/classificação , Humanos , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
In the progression of the life cycle of Plasmodium falciparum, a small proportion of asexual parasites differentiate into male or female sexual forms called gametocytes. Just like their asexual counterparts, gametocytes are contained within the infected host's erythrocytes (RBCs). However, unlike their asexual partners, they do not exit the RBC until they are taken up in a blood meal by a mosquito. In the mosquito midgut, they are stimulated to emerge from the RBC, undergo fertilization, and ultimately produce tens of thousands of sporozoites that are infectious to humans. This transmission cycle can be blocked by antibodies targeting proteins exposed on the parasite surface in the mosquito midgut, a process that has led to the development of candidate transmission-blocking vaccines (TBV), including some that are in clinical trials. Here we review the leading TBV antigens and highlight the ongoing search for additional gametocyte/gamete surface antigens, as well as antigens on the surfaces of gametocyte-infected erythrocytes, which can potentially become a new group of TBV candidates.
Assuntos
Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Animais , Humanos , Estágios do Ciclo de Vida , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/genética , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
An essential step in the transmission of the malaria parasite to the Anopheles vector is the transformation of the mature gametocytes into gametes in the mosquito gut, where they egress from the erythrocytes and mate to produce a zygote, which matures into a motile ookinete. Osmiophilic bodies are electron dense secretory organelles of the female gametocytes which discharge their contents during gamete formation, suggestive of a role in gamete egress. Only one protein with no functional annotation, Pfg377, is described to specifically reside in osmiophilic bodies in Plasmodium falciparum Importantly, Pfg377 defective gametocytes lack osmiophilic bodies and fail to infect mosquitoes, as confirmed here with newly produced pfg377 disrupted parasites. The unique feature of Pfg377 defective gametocytes of lacking osmiophilic bodies was here exploited to perform comparative, label free, global and affinity proteomics analyses of mutant and wild type gametocytes to identify components of these organelles. Subcellular localization studies with fluorescent reporter gene fusions and specific antibodies revealed an osmiophilic body localization for four out of five candidate gene products analyzed: the proteases PfSUB2 (subtilisin 2) and PfDPAP2 (Dipeptidyl aminopeptidase 2), the ortholog of the osmiophilic body component of the rodent malaria gametocytes PbGEST and a previously nonannotated 13 kDa protein. These results establish that osmiophilic bodies and their components are dispensable or marginally contribute (PfDPAP2) to gamete egress. Instead, this work reveals a previously unsuspected role of these organelles in P. falciparum development in the mosquito vector.
Assuntos
Organelas/metabolismo , Plasmodium falciparum/fisiologia , Proteômica/métodos , Proteínas de Protozoários/análise , Animais , Anopheles/parasitologia , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Feminino , Células Germinativas/metabolismo , Mutação , Proteínas de Protozoários/genética , Subtilisinas/metabolismoRESUMO
BACKGROUND: An effective malaria transmission-blocking vaccine (TBV) would be a major advance in the current efforts to eliminate and, ultimately, eradicate malaria. Antibodies against Plasmodium falciparum surface protein, Pfs25, are known to block parasite development in the mosquito vector. However, in initial clinical trials the limited immunogenicity of recombinant Pfs25 protein-in-adjuvant vaccines has been a challenge. METHODS: Novel human adenovirus type 5 (Ad5) vectors were used in heterologous prime boost vaccination strategies to augment the immune response against Pfs25. Specifically, an Ad5 vector that directs expression of full-length, membrane-bound Pfs25 was used as a priming immunization followed by a boost with Ad5 viral particles displaying only the Pfs25 epitope targeted by transmission-blocking antibodies 4B7 and 1D2 (Pfs25 aa 122-134) in hypervariable region 5 of the hexon capsid protein. RESULTS: This heterologous prime-boost vaccine strategy induced antibodies that significantly inhibit P. falciparum transmission to mosquitoes in a standard membrane-feeding assay. Further, immunized mice generated a robust anti-Pfs25 antibody response characterized by higher titer, higher relative avidity and a broader IgG subclass profile than observed with a homologous prime-boost with recombinant Pfs25/alum. CONCLUSION: The data suggest that focusing the immune response against defined epitopes displayed on the viral capsid is an effective strategy for transmission-blocking vaccine development.
Assuntos
Vacinas contra Adenovirus/genética , Anticorpos Antiprotozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/biossíntese , Epitopos/química , Vetores Genéticos , Células HEK293 , Células HeLa , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Malária Falciparum/transmissão , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/químicaRESUMO
BACKGROUND: Recent advances in malaria control efforts have led to an increased number of national malaria control programmes implementing pre-elimination measures and demonstrated the need to develop new tools to track and control malaria transmission. Key to understanding transmission is monitoring the prevalence and immune response against the sexual stages of the parasite, known as gametocytes, which are responsible for transmission. Sexual-stage specific antigens, Pfs230 and Pfs48/45, have been identified and shown to be targets for transmission blocking antibodies, but they have been difficult to produce recombinantly in the absence of a fusion partner. METHODS: Regions of Pfs48/45 and Pfs230 known to contain transmission blocking epitopes, 6C and C0, respectively, were produced in a Lactococcus lactis expression system and used in enzyme linked immunosorbent assays to determine the seroreactivity of 95 malaria patients living in the Central Region of Ghana. RESULTS: Pfs48/45.6C and Pfs230.C0 were successfully produced in L. lactis in the absence of a fusion partner using a simplified purification scheme. Seroprevalence for L. lactis-produced Pfs48/45.6C and Pfs230.C0 in the study population was 74.7 and 72.8%, respectively. CONCLUSIONS: A significant age-dependent increase in antibody titers was observed, which suggests a vaccine targeting these antigens could be boosted during a natural infection in the field.
Assuntos
Vacinas Antimaláricas/imunologia , Malária Falciparum/epidemiologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/imunologia , Adolescente , Adulto , Fatores Etários , Antígenos de Bactérias/análise , Criança , Pré-Escolar , Feminino , Gana/epidemiologia , Humanos , Lactente , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Estudos Soroepidemiológicos , Adulto JovemRESUMO
BACKGROUND: Blocking malaria transmission is an important step in eradicating malaria. In the field, transmission requires the production of sexual stage Plasmodium parasites, called gametocytes, which are not effectively killed by the commonly used anti-malarials allowing individuals to remain infectious after clearance of asexual parasites. METHODS: To identify new gametocytocidal compounds, a library of 45,056 compounds with diverse structures was screened using a high throughput gametocyte viability assay. The characteristics of active hits were further evaluated against asexual stage parasites in a growth inhibition assay. Their cytotoxicity were tested against mammalian cells in a cytotoxicity assay. The chemical scaffold similarity of active hits were studied using scaffold cluster analysis. RESULTS: A set of 23 compounds were identified and further confirmed for their activity against gametocytes. All the 23 confirmed compounds possess dual-activities against both gametocytes responsible for human to mosquito transmission and asexual parasites that cause the clinical symptoms. Three of these compounds were fourfold more active against gametocytes than asexual parasites. Further cheminformatic analysis revealed three sets of novel scaffolds, including highly selective 4-1H-pyrazol-5-yl piperidine analogs. CONCLUSIONS: This study revealed important new structural scaffolds that can be used as starting points for dual activity anti-malarial drug development.
Assuntos
Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/química , Antimaláricos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2 , Humanos , Concentração Inibidora 50 , Estrutura MolecularRESUMO
Novel imidazo[4,5-c]quinolin-2-ones were synthesized and evaluated in asexual blood stage and late stage gametocyte assays of Plasmodium falciparum, a major causative agent of malaria. The design of these compounds is based on a recently identified lead compound from a high throughput screen. A concise synthesis was developed that allowed for generation of analogues with substitution around both the quinoline and imidazolidinone rings. Through structure-activity relationship studies, a number of potent compounds were identified that possessed excellent antimalarial activity against both the asexual and sexual stages with minimal cytotoxicity in mammalian cells. This is the first Letter describing SAR and gametocytocidal activity of imidazo[4,5-c]quinolin-2-ones, a new lead series for malaria treatment and prevention.
Assuntos
Antimaláricos/farmacologia , Imidazóis/farmacologia , Malária/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Quinolonas/farmacologia , Antimaláricos/síntese química , Antimaláricos/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Hep G2 , Ensaios de Triagem em Larga Escala , Humanos , Imidazóis/síntese química , Imidazóis/química , Estrutura Molecular , Testes de Sensibilidade Parasitária , Quinolonas/síntese química , Quinolonas/química , Relação Estrutura-AtividadeRESUMO
Gametocytes are essential for Plasmodium transmission, but little is known about the mechanisms that lead to their formation. Using piggyBac transposon-mediated insertional mutagenesis, we screened for parasites that no longer form mature gametocytes, which led to the isolation of 29 clones (insertional gametocyte-deficient mutants) that fail to form mature gametocytes. Additional analysis revealed 16 genes putatively responsible for the loss of gametocytogenesis, none of which has been previously implicated in gametocytogenesis. Transcriptional profiling and detection of an early stage gametocyte antigen determined that a subset of these mutants arrests development at stage I or in early stage II gametocytes, likely representing genes involved in gametocyte maturation. The remaining mutants seem to arrest before formation of stage I gametocytes and may represent genes involved in commitment to the gametocyte lineage.
Assuntos
Elementos de DNA Transponíveis/genética , Gametogênese/genética , Genes de Protozoários/genética , Mutagênese/genética , Plasmodium falciparum/genética , Animais , Teste de Complementação Genética , Células Germinativas/metabolismo , Modelos Biológicos , Mutagênese Insercional/genética , Mutação/genética , Parasitos/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Malaria elimination efforts would benefit from vaccines that block transmission of Plasmodium falciparum gametocytes from humans to mosquitoes. A clear understanding of gametocyte-specific antibody responses in exposed populations could help determine whether transmission-blocking vaccines (TBV) would be boosted by natural gametocyte exposure, and also inform the development of serologic tools to monitor gametocyte exposure in populations targeted for malaria elimination. To this end, plasma was collected from Malian children and adults before and after the 6-month malaria season and probed against a microarray containing 1,204 P. falciparum proteins. Using publicly available proteomic data, we classified 91 proteins as gametocyte specific and 69 as proteins not expressed by gametocytes. The overall breadth and magnitude of gametocyte-specific IgG responses increased during the malaria season, although they were consistently lower than IgG responses to nongametocyte antigens. Notably, IgG specific for the TBV candidates Pfs48/45 and Pfs230 increased during the malaria season. In addition, IgGs specific for the gametocyte proteins Pfmdv1, Pfs16, PF3D7_1346400, and PF3D7_1024800 were detected in nearly all subjects, suggesting that seroconversion to these proteins may be a sensitive indicator of gametocyte exposure, although further studies are needed to determine the specificity and kinetics of these potential serologic markers. These findings suggest that TBV-induced immunity would be boosted through natural gametocyte exposure, and that antibody responses to particular antigens may reliably indicate gametocyte exposure.
Assuntos
Anticorpos Antiprotozoários/imunologia , Células Germinativas/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Malária Falciparum/parasitologia , Masculino , Plasmodium falciparum/química , Plasmodium falciparum/classificação , Plasmodium falciparum/genética , Proteômica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Adulto JovemRESUMO
New strategies targeting Plasmodium falciparum gametocytes, the sexual-stage parasites that are responsible for malaria transmission, are needed to eradicate this disease. Most commonly used antimalarials are ineffective against P. falciparum gametocytes, allowing patients to continue to be infectious for over a week after asexual parasite clearance. A recent screen for gametocytocidal compounds demonstrated that the carboxylic polyether ionophore maduramicin is active at low nanomolar concentrations against P. falciparum sexual stages. In this study, we showed that maduramicin has an EC50 (effective concentration that inhibits the signal by 50%) of 14.8 nM against late-stage gametocytes and significantly blocks in vivo transmission in a mouse model of malaria transmission. In contrast to other reported gametocytocidal agents, maduramicin acts rapidly in vitro, eliminating gametocytes and asexual schizonts in less than 12 h without affecting uninfected red blood cells (RBCs). Ring stage parasites are cleared by 24 h. Within an hour of drug treatment, 40% of the normally crescent-shaped gametocytes round up and become spherical. The number of round gametocytes increases to >60% by 2 h, even before a change in membrane potential as monitored by MitoProbe DiIC1 (5) is detectable. Maduramicin is not preferentially taken up by gametocyte-infected RBCs compared to uninfected RBCs, suggesting that gametocytes are more sensitive to alterations in cation concentration than RBCs. Moreover, the addition of 15.6 nM maduramicin enhanced the gametocytocidal activity of the pyrazoleamide PA21A050, which is a promising new antimalarial candidate associated with an increase in intracellular Na(+) concentration that is proposed to be due to inhibition of PfATP4, a putative Na(+) pump. These results underscore the importance of cation homeostasis in sexual as well as asexual intraerythrocytic-stage P. falciparum parasites and the potential of targeting this pathway for drug development.
Assuntos
Antimaláricos/farmacologia , Benzimidazóis/farmacologia , Lactonas/farmacologia , Malária/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Pirazóis/farmacologia , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Feminino , Gametogênese , Malária/transmissão , Camundongos Endogâmicos , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/patogenicidade , Esquizontes/efeitos dos fármacosRESUMO
Forty percent of the world's population is threatened by malaria, which is caused by Plasmodium parasites and results in an estimated 200 million clinical cases and 650,000 deaths each year. Drug resistance has been reported for all commonly used antimalarials and has prompted screens to identify new drug candidates. However, many of these new candidates have not been evaluated against the parasite stage responsible for transmission, gametocytes. If Plasmodium falciparum gametocytes are not eliminated, patients continue to spread malaria for weeks after asexual parasite clearance. Asymptomatic individuals can also harbor gametocyte burdens sufficient for transmission, and a safe, effective gametocytocidal agent could also be used in community-wide malaria control programs. Here, we identify 15 small molecules with nanomolar activity against late-stage gametocytes. Fourteen are diaminonaphthoquinones (DANQs), and one is a 2-imino-benzo[d]imidazole (IBI). One of the DANQs identified, SJ000030570, is a lead antimalarial candidate. In contrast, 94% of the 650 compounds tested are inactive against late-stage gametocytes. Consistent with the ineffectiveness of most approved antimalarials against gametocytes, of the 19 novel compounds with activity against known anti-asexual-stage targets, only 3 had any strong effect on gametocyte viability. These data demonstrate the distinct biology of the transmission stages and emphasize the importance of screening for gametocytocidal activity. The potent gametocytocidal activity of DANQ and IBI coupled with their efficacy against asexual parasites provides leads for the development of antimalarials with the potential to prevent both the symptoms and the spread of malaria.
Assuntos
Antimaláricos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Naftoquinonas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/química , Células Hep G2 , Humanos , Imidazóis/farmacologia , Naftoquinonas/química , Relação Estrutura-AtividadeRESUMO
The prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodium parasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novo via the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum, the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparum by assessing the functional consequences of deleting fabI and fabB/F. Targeted deletion or disruption of these genes in P. falciparum did not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodium parasites P. berghei and P. yoelii, where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anopheles mosquito midgut.
Assuntos
Anopheles/parasitologia , Ácidos Graxos/biossíntese , Insetos Vetores/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Esporozoítos/metabolismo , Animais , Trato Gastrointestinal/parasitologia , Humanos , Malária Falciparum/parasitologia , Oocistos/crescimento & desenvolvimento , Oocistos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Esporozoítos/crescimento & desenvolvimentoRESUMO
Malaria transmission requires the production of male and female gametocytes in the human host followed by fertilization and sporogonic development in the mosquito midgut. Although essential for the spread of malaria through the population, little is known about the initiation of gametocytogenesis in vitro or in vivo. Using a gametocyte-defective parasite line and genetic complementation, we show that Plasmodium falciparumgametocyte development 1 gene (Pfgdv1), encoding a peri-nuclear protein, is critical for early sexual differentiation. Transcriptional analysis of Pfgdv1 negative and positive parasite lines identified a set of gametocytogenesis early genes (Pfge) that were significantly down-regulated (>10 fold) in the absence of Pfgdv1 and expression was restored after Pfgdv1 complementation. Progressive accumulation of Pfge transcripts during successive rounds of asexual replication in synchronized cultures suggests that gametocytes are induced continuously during asexual growth. Comparison of Pfge gene transcriptional profiles in patient samples divided the genes into two groups differing in their expression in mature circulating gametocytes and providing candidates to evaluate gametocyte induction and maturation separately in vivo. The expression profile of one of the early gametocyte specific genes, Pfge1, correlated significantly with asexual parasitemia, which is consistent with the ongoing induction of gametocytogenesis during asexual growth observed in vitro and reinforces the need for sustained transmission-blocking strategies to eliminate malaria.
Assuntos
Genes de Protozoários , Malária/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Malária/transmissão , Parasitemia , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/sangue , Reprodução Assexuada , Transcrição Gênica , TranscriptomaRESUMO
In the current era of malaria eradication, reducing transmission is critical. Assessment of transmissibility requires tools that can accurately identify the various developmental stages of the malaria parasite, particularly those required for transmission (sexual stages). Here, we present a method for estimating relative amounts of Plasmodium falciparum asexual and sexual stages from gene expression measurements. These are modeled using constrained linear regression to characterize stage-specific expression profiles within mixed-stage populations. The resulting profiles were analyzed functionally by gene set enrichment analysis (GSEA), confirming differentially active pathways such as increased mitochondrial activity and lipid metabolism during sexual development. We validated model predictions both from microarrays and from quantitative RT-PCR (qRT-PCR) measurements, based on the expression of a small set of key transcriptional markers. This sufficient marker set was identified by backward selection from the whole genome as available from expression arrays, targeting one sentinel marker per stage. The model as learned can be applied to any new microarray or qRT-PCR transcriptional measurement. We illustrate its use in vitro in inferring changes in stage distribution following stress and drug treatment and in vivo in identifying immature and mature sexual stage carriers within patient cohorts. We believe this approach will be a valuable resource for staging lab and field samples alike and will have wide applicability in epidemiological studies of malaria transmission.
Assuntos
Expressão Gênica , Malária Falciparum/genética , Animais , Biomarcadores , Humanos , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Seleção GenéticaRESUMO
BACKGROUND: Plasmodium vivax preferentially infects Duffy-positive reticulocytes and infections typically have few parasite-infected cells in the peripheral circulation. These features complicate detection and quantification by flow cytometry (FC) using standard nucleic acid-based staining methods. A simple antibody-based FC method was developed for rapid parasite detection along with simultaneous detection of other parasite and erythrocyte markers. METHODS: Clinical samples were collected from patients diagnosed with P. vivax at a district Malaria Clinic in Kanchanaburi, Thailand. One µL of infected blood was washed, fixed, stained with a Plasmodium pan-specific anti-PfBiP antibody conjugated with Alexa Fluor 660, and analysed by FC. Additional primary conjugated antibodies for stage-specific markers of P. vivax for late trophozoite-early schizonts (MSP1-Alexa Fluor 660), late-stage schizonts (DBP-Alexa Fluor 555), and sexual stages (Pvs16) were used to differentiate intra-erythrocytic developmental stages. RESULTS: The percentages of P. vivax-infected cells determined by the FC method and manually by microscopic examination of Giemsa-stained thick blood smears were positively correlated by Spearman's rank correlation coefficient (R2=0.93843) from 0.001 to 1.00% P. vivax-infected reticulocytes. CONCLUSIONS: The FC-based method is a simple, robust, and efficient method for detecting P. vivax-infected reticulocytes.
Assuntos
Células Sanguíneas/parasitologia , Citometria de Fluxo/métodos , Malária Vivax/diagnóstico , Plasmodium vivax/isolamento & purificação , Anticorpos Antiprotozoários , Antígenos de Protozoários/análise , Corantes Fluorescentes/análise , Humanos , Coloração e Rotulagem , TailândiaRESUMO
BACKGROUND: Millions of individuals being treated for human immunodeficiency virus (HIV) live in malaria-endemic areas, but the effects of these treatments on malaria transmission are unknown. While drugs like HIV protease inhibitors (PIs) and trimethoprim-sulfamethoxazole (TMP-SMX) have known activity against parasites during liver or asexual blood stages, their effects on transmission stages require further study. METHODS: The HIV PIs lopinavir and saquinavir, the nonnucleoside reverse-transcriptase inhibitor nevirapine, and the antibiotic TMP-SMX were assessed for activity against Plasmodium falciparum transmission stages. The alamarBlue assay was used to determine the effects of drugs on gametocyte viability, and exflagellation was assessed to determine the effects of drugs on gametocyte maturation. The effects of drug on transmission were assessed by calculating the mosquito oocyst count as a marker for infectivity, using standard membrane feeding assays. RESULTS: Lopinavir and saquinavir have gametocytocidal and transmission blocking activities at or approaching clinically relevant treatment levels, while nevirapine does not. TMP-SMX is not gametocytocidal, but at prophylactic levels it blocks transmission. CONCLUSIONS: Specific HIV treatments have gametocyte killing and transmission-blocking effects. Clinical studies are warranted to evaluate these findings and their potential impact on eradication efforts.
Assuntos
Fármacos Anti-HIV/farmacologia , Antimaláricos/farmacologia , Inibidores da Protease de HIV/farmacologia , Lopinavir/farmacologia , Nevirapina/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Saquinavir/farmacologia , Combinação Trimetoprima e Sulfametoxazol/farmacologia , Animais , Anopheles/parasitologia , Relação Dose-Resposta a Droga , Humanos , Malária Falciparum/prevenção & controle , Malária Falciparum/transmissão , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/fisiologiaRESUMO
Resistance to clinical malaria takes years to develop even in hyperendemic regions and sterilizing immunity has rarely been observed. To evaluate the maturation of the host response against controlled repeat exposures to P. falciparum (Pf) NF54 strain-infected mosquitoes, we systematically monitored malaria-naïve participants through an initial exposure to uninfected mosquitoes and 4 subsequent homologous exposures to Pf-infected mosquitoes over 21 months (n = 8 males) (ClinicalTrials.gov# NCT03014258). The primary outcome was to determine whether protective immunity against parasite infection develops following repeat CHMI and the secondary outcomes were to track the clinical signs and symptoms of malaria and anti-Pf antibody development following repeat CHMI. After two exposures, time to blood stage patency increases significantly and the number of reported symptoms decreases indicating the development of clinical tolerance. The time to patency correlates positively with both anti-Pf circumsporozoite protein (CSP) IgG and CD8 + CD69+ effector memory T cell levels consistent with partial pre-erythrocytic immunity. IFNγ levels decrease significantly during the participants' second exposure to high blood stage parasitemia and could contribute to the decrease in symptoms. In contrast, CD4-CD8 + T cells expressing CXCR5 and the inhibitory receptor, PD-1, increase significantly after subsequent Pf exposures, possibly dampening the memory response and interfering with the generation of robust sterilizing immunity.