RESUMO
The active metabolites of primaquine, in particular 5-hydroxyprimaquine, likely responsible for the clearance of dormant hypnozoites, are produced through the hepatic CYP450 2D6 (CYP2D6) enzymatic pathway. With the inherent instability of 5-hydroxyprimaquine, a stable surrogate, 5,6-orthoquinone, can now be detected and measured in the urine as part of primaquine pharmacokinetic studies. This study performed CYP450 2D6 genotyping and primaquine pharmacokinetic testing, to include urine 5,6-orthoquinone, in 27 healthy adult Cambodians, as a preliminary step to prepare for future clinical studies assessing primaquine efficacy for Plasmodium vivax infections. The CYP2D6 *10 reduced activity allele was found in 57% of volunteers, and the CYP2D6 genotypes were dominated by *1/*10 (33%) and *10/*10 (30%). Predicted phenotypes were evenly split between Normal Metabolizer (NM) and Intermediate Metabolizer (IM) except for one volunteer with a gene duplication and unclear phenotype, classifying as either IM or NM. Median plasma primaquine (PQ) area under the curve (AUC) was lower in the NM group (460 h*ng/mL) compared to the IM group (561 h*ng/mL), although not statistically significant. Similar to what has been found in the US study, no 5,6-orthoquinone was detected in the plasma. The urine creatinine-corrected 5,6-orthoquinone AUC in the NM group was almost three times higher than in the IM group, with peak measurements (Tmax) at 4 h. Although there is variation among individuals, future studies examining the relationship between the levels of urine 5,6-orthoquinone and primaquine radical cure efficacy could result in a metabolism biomarker predictive of radical cure.
Assuntos
Antimaláricos , Malária Vivax , Adulto , Antimaláricos/farmacocinética , Antimaláricos/uso terapêutico , Povo Asiático , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Humanos , Malária Vivax/tratamento farmacológico , Plasmodium vivax/genética , Primaquina/análogos & derivados , Primaquina/farmacocinética , Primaquina/uso terapêuticoRESUMO
BACKGROUND: The rise in Plasmodium falciparum resistance to dihydroartemisinin-piperaquine (DHA-PPQ) treatment has been documented in the Greater Mekong Subregion with associations with mutations in the P. falciparum chloroquine resistance transporter (pfcrt) and plasmepsin 2 (pfpm2) genes. However, it is unclear whether other genes also play a role with PPQ resistance, such as the E415G mutation in the exonuclease (pfexo) gene. The aim of this study was to investigate the role of this mutation in PPQ resistance by generating transgenic parasites expressing the pfexo-E415G mutant allele. METHODS: Transgenic parasite clones carrying the E415G mutation in PfEXO of the B5 isolate were derived by CRISPR-Cas9 gene editing and verified using PCR and gene sequencing. Polymorphisms of pfkelch-13, pfcrt, and pfexo were examined by PCR while the copy number variations of pfpm2 were examined by both relative quantitative real-time PCR and the duplication breakpoint assay. Drug sensitivity against a panel of antimalarials, the ring-stage survival assay (RSA), the PPQ survival assay (PSA), and bimodal dose-response curves were used to evaluate antimalarial susceptibility. RESULTS: The transgenic line, B5-rexo-E415G-B8, was successfully generated. The PPQ-IC90, %PPQ survival, and the bimodal dose-response clearly showed that E415G mutation in PfEXO of B5 isolate remained fully susceptible to PPQ. Furthermore, growth assays demonstrated that the engineered parasites grew slightly faster than the unmodified parental isolates whereas P. falciparum isolates harbouring pfkelch-13, pfcrt, and pfexo mutations with multiple copies of pfpm2 grew much more slowly. CONCLUSIONS: Insertion of the E415G mutation in PfEXO did not lead to increased PPQ-IC90 and %PPQ survival, suggesting that this mutation alone may not be associated with PPQ resistance, but could still be an important marker if used in conjunction with other markers for monitoring PPQ-resistant parasites. The results also highlight the importance of monitoring and evaluating suspected genetic mutations with regard to parasite fitness and resistance.
Assuntos
Antimaláricos , Malária Falciparum , Parasitos , Quinolinas , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Variações do Número de Cópias de DNA , Resistência a Medicamentos/genética , Exonucleases/genética , Exonucleases/farmacologia , Exonucleases/uso terapêutico , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Mutação , Fosfodiesterase I/genética , Fosfodiesterase I/farmacologia , Piperazinas , Plasmodium falciparum , Mutação Puntual , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Quinolinas/farmacologia , Quinolinas/uso terapêuticoRESUMO
BACKGROUND: While human cases of Plasmodium knowlesi are now regularly recognized in Southeast Asia, infections with other simian malaria species, such as Plasmodium cynomolgi, are still rare. There has been a handful of clinical cases described, all from Malaysia, and retrospective studies of archived blood samples in Thailand and Cambodia have discovered the presence P. cynomolgi in isolates using polymerase chain reaction (PCR) assays. CASE PRESENTATION: In Thailand, an ongoing malaria surveillance study enrolled two patients from Yala Province diagnosed with Plasmodium vivax by blood smear, but who were subsequently found to be negative by PCR. Expanded PCR testing of these isolates detected mono-infection with P. cynomolgi, the first time this has been reported in Thailand. Upon re-testing of 60 isolates collected from Yala, one other case was identified, a co-infection of P. cynomolgi and P. vivax. The clinical course for all three was relatively mild, with symptoms commonly seen in malaria: fever, chills and headaches. All infections were cured with a course of chloroquine and primaquine. CONCLUSION: In malaria-endemic areas with macaque populations, cases of simian malaria in humans are being reported at an increasing rate, although still comprise a very small percentage of total cases. Plasmodium cynomolgi and P. vivax are challenging to distinguish by blood smear; therefore, PCR can be employed when infections are suspected or as part of systematic malaria surveillance. As Thai MoPH policy schedules regular follow-up visits after each malaria infection, identifying those with P. cynomolgi will allow for monitoring of treatment efficacy, although at this time P. cynomolgi appears to have an uncomplicated clinical course and good response to commonly used anti-malarials.
Assuntos
Malária Vivax , Malária , Parasitos , Plasmodium cynomolgi , Plasmodium knowlesi , Animais , Humanos , Malária/diagnóstico , Malária/tratamento farmacológico , Malária/epidemiologia , Malária Vivax/diagnóstico , Malária Vivax/tratamento farmacológico , Malária Vivax/epidemiologia , Estudos Retrospectivos , Tailândia/epidemiologiaRESUMO
Human papillomavirus (HPV) is one of the most common sexually transmitted infections in men and women. Most HPV studies have focused on vaccination toward women to prevent consequences of developing cervical cancer. However, persistent infections can cause penile, anal, and oropharyngeal cancers in men. Therefore, recent public health recommendations toward vaccination in men have been raised. There is limited HPV prevalence data among men in many countries, including Thailand. We conducted HPV sera IgG ELISA testing on a repository sera of Thai men (average age 21 years old) entering the Royal Thai Army as recruits in 2013 (n = 1000). HPV IgG antibodies against virus-like particles of HPV- type 6, 11, 16e, and 18 were evaluated using a commercial ELISA kit. Overall, the anti-HPV IgG was 47% (95% CI: 44%-50%). HPV seroprevalence was significantly associated with residence regions with the highest prevalence in South (64%), but not associated with educational level, marital status, or type of residence. This data suggested that almost half of the Thai men in this cohort were exposed to HPV by the age of 21. Thus, HPV vaccination provided to male adolescents should be considered for disease prevention and minimizing transmission to sexual partners.
Assuntos
Alphapapillomavirus , Infecções por Papillomavirus , Adolescente , Adulto , Feminino , Humanos , Masculino , Papillomaviridae , Infecções por Papillomavirus/epidemiologia , Estudos Soroepidemiológicos , Tailândia/epidemiologia , Adulto JovemRESUMO
BACKGROUND: Newly emerged mutations within the Plasmodium falciparum chloroquine resistance transporter (PfCRT) can confer piperaquine resistance in the absence of amplified plasmepsin II (pfpm2). In this study, we estimated the prevalence of co-circulating piperaquine resistance mutations in P. falciparum isolates collected in northern Cambodia from 2009 to 2017. METHODS: The sequence of pfcrt was determined for 410 P. falciparum isolates using PacBio amplicon sequencing or whole genome sequencing. Quantitative polymerase chain reaction was used to estimate pfpm2 and pfmdr1 copy number. RESULTS: Newly emerged PfCRT mutations increased in prevalence after the change to dihydroartemisinin-piperaquine in 2010, with >98% of parasites harboring these mutations by 2017. After 2014, the prevalence of PfCRT F145I declined, being outcompeted by parasites with less resistant, but more fit PfCRT alleles. After the change to artesunate-mefloquine, the prevalence of parasites with amplified pfpm2 decreased, with nearly half of piperaquine-resistant PfCRT mutants having single-copy pfpm2. CONCLUSIONS: The large proportion of PfCRT mutants that lack pfpm2 amplification emphasizes the importance of including PfCRT mutations as part of molecular surveillance for piperaquine resistance in this region. Likewise, it is critical to monitor for amplified pfmdr1 in these PfCRT mutants, as increased mefloquine pressure could lead to mutants resistant to both drugs.
Assuntos
Antimaláricos/farmacologia , Biomarcadores/metabolismo , Resistência a Medicamentos/genética , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Piperazinas/uso terapêutico , Proteínas de Protozoários/genética , Quinolinas/uso terapêutico , Animais , Antimaláricos/uso terapêutico , Camboja/epidemiologia , Resistência a Medicamentos/efeitos dos fármacos , Malária Falciparum/epidemiologia , Mefloquina/uso terapêutico , Mutação/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Prevalência , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: A previous RTS,S/AS01B vaccine challenge trial demonstrated that a 3-dose (0-1-7-month) regimen with a fractional third dose can produce high vaccine efficacy (VE) in adults challenged 3 weeks after vaccination. This study explored the VE of different delayed fractional dose regimens of adult and pediatric RTS,S/AS01 formulations. METHODS: A total of 130 participants were randomized into 5 groups. Four groups received 3 doses of RTS,S/AS01B or RTS,S/AS01E on a 0-1-7-month schedule, with the final 1 or 2 doses being fractional (one-fifth dose volume). One group received 1 full (month 0) and 1 fractional (month 7) dose of RTS,S/AS01E. Immunized and unvaccinated control participants underwent Plasmodium falciparum-infected mosquito challenge (controlled human malaria infection) 3 months after immunization, a timing chosen to potentially discriminate VEs between groups. RESULTS: The VE of 3-dose formulations ranged from 55% (95% confidence interval, 27%-72%) to 76% (48%-89%). Groups administered equivalent formulations of RTS,S/AS01E and RTS,S/AS01B demonstrated comparable VE. The 2-dose group demonstrated lower VE (29% [95% confidence interval, 6%-46%]). All regimens were well tolerated and immunogenic, with trends toward higher anti-circumsporozoite antibody titers in participants protected against infection. CONCLUSIONS: RTS,S/AS01E can provide VE comparable to an equivalent RTS,S/AS01B regimen in adults, suggesting a universal formulation may be considered. Results also suggest that the 2-dose regimen is inferior to the 3-dose regimens evaluated. CLINICAL TRIAL REGISTRATION: NCT03162614.
Assuntos
Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/imunologia , Malária/imunologia , Malária/prevenção & controle , Adolescente , Adulto , Feminino , Humanos , Esquemas de Imunização , Controle de Infecções , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Masculino , Pessoa de Meia-Idade , Plasmodium falciparum/imunologia , Vacinação , Adulto JovemRESUMO
BACKGROUND: High rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. The genetic markers plasmepsin 2 (pfpm2), exonuclease (pfexo) and chloroquine resistance transporter (pfcrt) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy. METHODS: To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia, and the presence of E415G-Exo and pfcrt mutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well as pfpm2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly cloned P. falciparum Cambodian isolates was also determined. RESULTS: The characterization of culture-adapted isolates revealed that the presence of novel pfcrt mutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence of pfpm2 amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose-response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovaquone-proguanil combinations revealed synergistic antimalarial activity. CONCLUSIONS: Surveillance for PPQ resistance in regions relying on DHA-PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance. P. falciparum harbouring novel pfcrt mutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence of pfpm2 amplification was not required to render parasites PPQ resistant suggesting that the increase in pfpm2 copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations against P. falciparum strains and when down-selecting novel drug candidates.
Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos , Genótipo , Fenótipo , Plasmodium falciparum/genética , Quinolinas/farmacologia , Camboja , Marcadores Genéticos , Plasmodium falciparum/efeitos dos fármacosRESUMO
BACKGROUND: Plasmodium vivax malaria requires a 2-week course of primaquine (PQ) for radical cure. Evidence suggests that the hepatic isoenzyme cytochrome P450 2D6 (CYP2D6) is the key enzyme required to convert PQ into its active metabolite. METHODS: CYP2D6 genotypes and phenotypes of 550 service personnel were determined, and the pharmacokinetics (PK) of a 30-mg oral dose of PQ was measured in 45 volunteers. Blood and urine samples were collected, with PQ and metabolites were measured using ultraperformance liquid chromatography with mass spectrometry. RESULTS: Seventy-six CYP2D6 genotypes were characterized for 530 service personnel. Of the 515 personnel for whom a single phenotype was predicted, 58% had a normal metabolizer (NM) phenotype, 35% had an intermediate metabolizer (IM) phenotype, 5% had a poor metabolizer (PM) phenotype, and 2% had an ultrametabolizer phenotype. The median PQ area under the concentration time curve from 0 to ∞ was lower for the NM phenotype as compared to the IM or PM phenotypes. The novel 5,6-ortho-quinone was detected in urine but not plasma from all personnel with the NM phenotype. CONCLUSION: The plasma PK profile suggests PQ metabolism is decreased in personnel with the IM or PM phenotypes as compared to those with the NM phenotype. The finding of 5,6-ortho-quinone, the stable surrogate for the unstable 5-hydroxyprimaquine metabolite, almost exclusively in personnel with the NM phenotype, compared with sporadic or no production in those with the IM or PM phenotypes, provides further evidence for the role of CYP2D6 in radical cure. CLINICAL TRIALS REGISTRATION: NCT02960568.
Assuntos
Antimaláricos/metabolismo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Genótipo , Primaquina/metabolismo , Administração Oral , Adolescente , Adulto , Antimaláricos/administração & dosagem , Antimaláricos/farmacocinética , Análise Química do Sangue , Cromatografia Líquida de Alta Pressão , Estudos de Coortes , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Militares , Fenótipo , Plasma/química , Primaquina/administração & dosagem , Primaquina/farmacocinética , Estados Unidos , Urinálise , Urina/química , Adulto JovemRESUMO
The South Pacific countries Solomon Islands, Vanuatu, and Papua New Guinea (PNG) adopted artemisinin-based combination therapies (ACTs) in 2008. We examined Kelch 13 and Kelch 12 genes in parasites originating from these countries before or at ACT introduction. Four Kelch 13 and two Kelch 12 novel sequence polymorphisms, not associated with artemisinin resistance, were observed in parasites from Solomon Islands and Vanuatu. No polymorphisms were observed in PNG parasites. The findings provide useful baseline information.
Assuntos
Genes de Protozoários/genética , Parasitos/genética , Plasmodium falciparum/genética , Plasmodium vivax/genética , Polimorfismo Genético/genética , Animais , Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Resistência a Medicamentos/genética , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Melanesia , Parasitos/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium vivax/efeitos dos fármacosRESUMO
False-negative results for Plasmodium falciparum histidine-rich protein (HRP) 2-based rapid diagnostic tests (RDTs) are increasing in Eritrea. We investigated HRP gene 2/3 (pfhrp2/pfhrp3) status in 50 infected patients at 2 hospitals. We showed that 80.8% (21/26) of patients at Ghindae Hospital and 41.7% (10/24) at Massawa Hospital were infected with pfhrp2-negative parasites and 92.3% (24/26) of patients at Ghindae Hospital and 70.8% (17/24) at Massawa Hospital were infected with pfhrp3-negative parasites. Parasite densities between pfhrp2-positive and pfhrp2-negative patients were comparable. All pfhrp2-negative samples had no detectable HRP2/3 antigen and showed negative results for HRP2-based RDTs. pfhrp2-negative parasites were genetically less diverse and formed 2 clusters with no close relationships to parasites from Peru. These parasites probably emerged independently by selection in Eritrea. High prevalence of pfhrp2-negative parasites caused a high rate of false-negative results for RDTs. Determining prevalence of pfhrp2-negative parasites is urgently needed in neighboring countries to assist case management policies.
Assuntos
Antígenos de Protozoários/genética , Deleção de Genes , Malária Falciparum/prevenção & controle , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Adolescente , Adulto , Idoso , Criança , Eritreia/epidemiologia , Variação Genética , Genótipo , Geografia , Humanos , Malária Falciparum/diagnóstico , Malária Falciparum/epidemiologia , Repetições de Microssatélites , Pessoa de Meia-Idade , Programas Nacionais de Saúde , Vigilância da População , Adulto JovemRESUMO
BACKGROUND: Non-human primates, such as the rhesus macaques, are the preferred model for down-selecting human malaria vaccine formulations, but the rhesus model is expensive and does not allow for direct efficacy testing of human malaria vaccines. Transgenic rodent parasites expressing genes of human Plasmodium are now routinely used for efficacy studies of human malaria vaccines. Mice have however rarely predicted success in human malaria trials and there is scepticism whether mouse studies alone are sufficient to move a vaccine candidate into the clinic. METHODS: A comparison of immunogenicity, fine-specificity and functional activity of two Alum-adjuvanted Plasmodium falciparum circumsporozoite protein (CSP)-based vaccines was conducted in mouse and rhesus models. One vaccine was a soluble recombinant protein (CSP) and the other was the same CSP covalently conjugated to the Qß phage particle (Qß-CSP). RESULTS: Mice showed different kinetics of antibody responses and different sensitivity to the NANP-repeat and N-terminal epitopes as compared to rhesus. While mice failed to discern differences between the protective efficacy of CSP versus Qß-CSP vaccine following direct challenge with transgenic Plasmodium berghei parasites, rhesus serum from the Qß-CSP-vaccinated animals induced higher in vivo sporozoite neutralization activity. CONCLUSIONS: Despite some immunologic parallels between models, these data demonstrate that differences between the immune responses induced in the two models risk conflicting decisions regarding potential vaccine utility in humans. In combination with historical observations, the data presented here suggest that although murine models may be useful for some purposes, non-human primate models may be more likely to predict the human response to investigational vaccines.
Assuntos
Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Animais , Feminino , Imunogenicidade da Vacina , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/imunologiaRESUMO
BACKGROUND: Models of controlled human malaria infection (CHMI) initiated by mosquito bite have been widely used to assess efficacy of preerythrocytic vaccine candidates in small proof-of-concept phase 2a clinical trials. Efficacy testing of blood-stage malaria parasite vaccines, however, has generally relied on larger-scale phase 2b field trials in malaria-endemic populations. We report the use of a blood-stage P. falciparum CHMI model to assess blood-stage vaccine candidates, using their impact on the parasite multiplication rate (PMR) as the primary efficacy end point. METHODS: Fifteen healthy United Kingdom adult volunteers were vaccinated with FMP2.1, a protein vaccine that is based on the 3D7 clone sequence of apical membrane antigen 1 (AMA1) and formulated in Adjuvant System 01 (AS01). Twelve vaccinees and 15 infectivity controls subsequently underwent blood-stage CHMI. Parasitemia was monitored by quantitative real-time polymerase chain reaction (PCR) analysis, and PMR was modeled from these data. RESULTS: FMP2.1/AS01 elicited anti-AMA1 T-cell and serum antibody responses. Analysis of purified immunoglobulin G showed functional growth inhibitory activity against P. falciparum in vitro. There were no vaccine- or CHMI-related safety concerns. All volunteers developed blood-stage parasitemia, with no impact of the vaccine on PMR. CONCLUSIONS: FMP2.1/AS01 demonstrated no efficacy after blood-stage CHMI. However, the model induced highly reproducible infection in all volunteers and will accelerate proof-of-concept testing of future blood-stage vaccine candidates. CLINICAL TRIALS REGISTRATION: NCT02044198.
Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Proteínas de Membrana/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adulto , ELISPOT , Eritrócitos/parasitologia , Feminino , Humanos , Imunogenicidade da Vacina , Estágios do Ciclo de Vida , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Plasmodium falciparum/fisiologia , Adulto JovemRESUMO
BACKGROUND: Three full doses of RTS,S/AS01 malaria vaccine provides partial protection against controlled human malaria parasite infection (CHMI) and natural exposure. Immunization regimens, including a delayed fractional third dose, were assessed for potential increased protection against malaria and immunologic responses. METHODS: In a phase 2a, controlled, open-label, study of healthy malaria-naive adults, 16 subjects vaccinated with a 0-, 1-, and 2-month full-dose regimen (012M) and 30 subjects who received a 0-, 1-, and 7-month regimen, including a fractional third dose (Fx017M), underwent CHMI 3 weeks after the last dose. Plasmablast heavy and light chain immunoglobulin messenger RNA sequencing and antibody avidity were evaluated. Protection against repeat CHMI was evaluated after 8 months. RESULTS: A total of 26 of 30 subjects in the Fx017M group (vaccine efficacy [VE], 86.7% [95% confidence interval [CI], 66.8%-94.6%]; P < .0001) and 10 of 16 in the 012M group (VE, 62.5% [95% CI, 29.4%-80.1%]; P = .0009) were protected against infection, and protection differed between schedules (P = .040, by the log rank test). The fractional dose boosting increased antibody somatic hypermutation and avidity and sustained high protection upon rechallenge. DISCUSSIONS: A delayed third fractional vaccine dose improved immunogenicity and protection against infection. Optimization of the RTS,S/AS01 immunization regimen may lead to improved approaches against malaria. CLINICAL TRIALS REGISTRATION: NCT01857869.
Assuntos
Esquemas de Imunização , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Adolescente , Adulto , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Afinidade de Anticorpos , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Recent vaccine studies have shown that the magnitude of an antibody response is often insufficient to explain efficacy, suggesting that characteristics regarding the quality of the antibody response, such as its fine specificity and functional activity, may play a major role in protection. Previous studies of the lead malaria vaccine candidate, RTS,S, have shown that circumsporozoite protein (CSP)-specific antibodies and CD4(+) T cell responses are associated with protection, however the role of fine specificity and biological function of CSP-specific antibodies remains to be elucidated. Here, the relationship between fine specificity, opsonization-dependent phagocytic activity and protection in RTS,S-induced antibodies is explored. METHODS: A new method for measuring the phagocytic activity mediated by CSP-specific antibodies in THP-1 cells is presented and applied to samples from a recently completed phase 2 RTS,S/AS01 clinical trial. The fine specificity of the antibody response was assessed using ELISA against three antigen constructs of CSP: the central repeat region, the C-terminal domain and the full-length protein. A multi-parameter analysis of phagocytic activity and fine-specificity data was carried out to identify potential correlates of protection in RTS,S. RESULTS: Results from the newly developed assay revealed that serum samples from RTS,S recipients displayed a wide range of robust and repeatable phagocytic activity. Phagocytic activity was correlated with full-length CSP and C-terminal specific antibody titres, but not to repeat region antibody titres, suggesting that phagocytic activity is primarily driven by C-terminal antibodies. Although no significant difference in overall phagocytic activity was observed with respect to protection, phagocytic activity expressed as 'opsonization index', a relative measure that normalizes phagocytic activity with CS antibody titres, was found to be significantly lower in protected subjects than non-protected subjects. CONCLUSIONS: Opsonization index was identified as a surrogate marker of protection induced by the RTS,S/AS01 vaccine and determined how antibody fine specificity is linked to opsonization activity. These findings suggest that the role of opsonization in protection in the RTS,S vaccine may be more complex than previously thought, and demonstrate how integrating multiple immune measures can provide insight into underlying mechanisms of immunity and protection.
Assuntos
Anticorpos Antiprotozoários/sangue , Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Proteínas Opsonizantes/sangue , Fagocitose , Vacinas Sintéticas/imunologia , Linhagem Celular , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Humanos , Vacinas Antimaláricas/administração & dosagem , Vacinas Sintéticas/administração & dosagemRESUMO
Artemisinin-induced dormancy is a proposed mechanism for failures of monotherapy and is linked with artemisinin resistance in Plasmodium falciparum. The biological characterization and dynamics of dormant parasites are not well understood. Here we report that after dihydroartemisinin treatment in vitro, a small subset of morphologically dormant parasites was stained with rhodamine 123 (RH), a mitochondrial membrane potential marker, and persisted to recovery. RH-positive parasites sorted with fluorescence-activated cell sorting resumed growth at 10,000/well whereas RH-negative parasites failed to recover at 5 million/well. Furthermore, transcriptional activity for mitochondrial enzymes was detected only in RH-positive dormant parasites. Importantly, after treatment of dormant parasites with different concentrations of atovaquone, a mitochondrial inhibitor, the recovery of dormant parasites was delayed or stopped. This demonstrates that mitochondrial activity is critical for survival and regrowth of dormant parasites and that RH staining provides a means of identifying these parasites. These findings provide novel paths for studying and eradicating this dormant stage.
Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/fisiologia , Atovaquona/farmacologia , Corantes Fluorescentes/análise , Genes Mitocondriais , Humanos , Rodamina 123/análiseRESUMO
Artemisinin (ART)-based combination therapy (ACT) is used as the first-line treatment of uncomplicated falciparum malaria worldwide. However, despite high potency and rapid action, there is a high rate of recrudescence associated with ART monotherapy or ACT long before the recent emergence of ART resistance. ART-induced ring-stage dormancy and recovery have been implicated as possible causes of recrudescence; however, little is known about the characteristics of dormant parasites, including whether dormant parasites are metabolically active. We investigated the transcription of 12 genes encoding key enzymes in various metabolic pathways in P. falciparum during dihydroartemisinin (DHA)-induced dormancy and recovery. Transcription analysis showed an immediate downregulation for 10 genes following exposure to DHA but continued transcription of 2 genes encoding apicoplast and mitochondrial proteins. Transcription of several additional genes encoding apicoplast and mitochondrial proteins, particularly of genes encoding enzymes in pyruvate metabolism and fatty acid synthesis pathways, was also maintained. Additions of inhibitors for biotin acetyl-coenzyme A (CoA) carboxylase and enoyl-acyl carrier reductase of the fatty acid synthesis pathways delayed the recovery of dormant parasites by 6 and 4 days, respectively, following DHA treatment. Our results demonstrate that most metabolic pathways are downregulated in DHA-induced dormant parasites. In contrast, fatty acid and pyruvate metabolic pathways remain active. These findings highlight new targets to interrupt recovery of parasites from ART-induced dormancy and to reduce the rate of recrudescence following ART treatment.
Assuntos
Ácidos Graxos/biossíntese , Estágios do Ciclo de Vida/genética , Proteínas Mitocondriais/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Piruvatos/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Antimaláricos/farmacologia , Apicoplastos/efeitos dos fármacos , Apicoplastos/genética , Apicoplastos/metabolismo , Artemisininas/farmacologia , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/antagonistas & inibidores , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/genética , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Inibidores Enzimáticos/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Regulação da Expressão Gênica , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Transcrição GênicaRESUMO
BACKGROUND: Chloroquine (CQ), alone or in combination with sulphadoxine-pyrimethamine, was widely used for the treatment of Plasmodium falciparum and Plasmodium vivax for several decades in both Vanuatu and Solomon Islands prior to the introduction of artemether-lumefantrine (AL) in 2008. However, the effect of chloroquine selection on parasite population, which may affect the efficacy of lumefantrine or other partner drugs of artemisinin, has not been well assessed. This study aims to provide baseline data on molecular markers (pfcrt and pfmdr1), along with the origins of pfcrt, prior to the introduction of AL. METHODS: Blood spots were obtained from epidemiological surveys conducted on Tanna Island, Tafea Province, Vanuatu and Temotu Province, Solomon Islands in 2008. Additional samples from Malaita Province, Solomon Islands were collected as part of an artemether-lumefantrine efficacy study in 2008. Plasmodium falciparum pfcrt and pfmdr1 genes were examined for polymorphisms. Microsatellite markers flanking pfcrt were also examined to ascertain origins of CQ resistance. RESULTS: Pfcrt analysis revealed 100% of parasites from Tafea Province, Vanuatu and Malaita Province, Solomon Islands and 98% of parasites from Temotu Province, Solomon Islands carried the K76T polymorphism that confers CQ resistance. Comparison of pfcrt allelic patterns and microsatellite markers flanking pfcrt revealed six haplotypes with more than 70% of isolates possessing haplotypes very similar to those observed in Papua New Guinea. The dominant (98.5%) pfmdr1 allele across all island groups was YYCND. CONCLUSIONS: Prior to the introduction of AL in the Solomon Islands and Vanuatu, P. falciparum isolates possessed point mutations known to confer CQ resistance and possibly associated with a decreased susceptibility to quinine and halofantrine, but an increased susceptibility to artemisinin and lumefantrine. Overall, pfcrt allelic types and the flanking microsatellite markers exhibited similarities to those of Papua New Guinea, suggesting these parasites share a common ancestry. The current use of AL for both P. falciparum and P. vivax infections will enable changes in these markers, in the absence of CQ pressure, to be monitored.
Assuntos
Antimaláricos/uso terapêutico , Artemisininas/uso terapêutico , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Criança , Pré-Escolar , Teste em Amostras de Sangue Seco , Quimioterapia Combinada , Haplótipos , Humanos , Malária Falciparum/epidemiologia , Melanesia/epidemiologia , Repetições de Microssatélites , Prevalência , Vanuatu/epidemiologiaRESUMO
BACKGROUND: Plasmodium falciparum and Plasmodium vivax are endemic in Vanuatu and the Solomon Islands. While both countries have introduced artemether-lumefantrine (AL) as first-line therapy for both P. falciparum and P. vivax since 2008, chloroquine and sulphadoxine-pyrimethamine (SP) were used as first-line therapy for many years prior to the introduction of AL. Limited data are available on the extent of SP resistance at the time of policy change. METHODS: Blood spots were obtained from epidemiological surveys conducted on Tanna Island, Tafea Province, Vanuatu and Temotu Province, Solomon Islands in 2008. Additional samples from Malaita Province, Solomon Islands were collected as part of an AL therapeutic efficacy study conducted in 2008. Plasmodium vivax and P. falciparum dhfr and dhps genes were sequenced to detect nucleotide polymorphisms. RESULTS: All P. falciparum samples analysed (n=114) possessed a double mutant pfdhfr allele (C59R/S108N). Additionally, mutation A437G in pfhdps was detected in a small number of samples 2/13, 1/17 and 3/26 from Tanna Island, Vanuatu and Temotu and Malaita Provinces Solomon Islands respectively. Mutations were also common in pvdhfr from Tanna Island, Vanuatu, where 33/51 parasites carried the double amino acid substitution S58R/S117N, while in Temotu and Malaita Provinces, Solomon Islands 32/40 and 39/46 isolates carried the quadruple amino acid substitution F57L/S58R/T61M/S117T in DHFR respectively. No mutations in pvdhps (n=108) were detected in these three island groups. CONCLUSION: Prior to the introduction of AL, there was a moderate level of SP resistance in the P. falciparum population that may cause SP treatment failure in young children. Of the P. vivax isolates, a majority of Solomon Islands isolates carried quadruple mutant pvdhfr alleles while a majority of Vanuatu isolates carried double mutant pvdhfr alleles. This suggests a higher level of SP resistance in the P. vivax population in Solomon Islands compared to the sympatric P. falciparum population and there is a higher level of SP resistance in P. vivax parasites from Solomon Islands than Vanuatu. This study demonstrates that the change of treatment policy in these countries from SP to ACT was timely. The information also provides a baseline for future monitoring.
Assuntos
Di-Hidropteroato Sintase/genética , Plasmodium falciparum/genética , Plasmodium vivax/genética , Tetra-Hidrofolato Desidrogenase/genética , Antimaláricos , Artemisininas , Estudos Transversais , Teste em Amostras de Sangue Seco , Combinação de Medicamentos , Quimioterapia Combinada , Marcadores Genéticos , Humanos , Malária/epidemiologia , Malária/parasitologia , Melanesia/epidemiologia , Mutação/genética , Plasmodium falciparum/enzimologia , Plasmodium vivax/enzimologia , Pirimetamina , Sulfadoxina , Vanuatu/epidemiologiaRESUMO
Malaria genomic surveillance often estimates parasite genetic relatedness using metrics such as Identity-By-Decent (IBD), yet strong positive selection stemming from antimalarial drug resistance or other interventions may bias IBD-based estimates. In this study, we use simulations, a true IBD inference algorithm, and empirical data sets from different malaria transmission settings to investigate the extent of this bias and explore potential correction strategies. We analyze whole genome sequence data generated from 640 new and 3089 publicly available Plasmodium falciparum clinical isolates. We demonstrate that positive selection distorts IBD distributions, leading to underestimated effective population size and blurred population structure. Additionally, we discover that the removal of IBD peak regions partially restores the accuracy of IBD-based inferences, with this effect contingent on the population's background genetic relatedness and extent of inbreeding. Consequently, we advocate for selection correction for parasite populations undergoing strong, recent positive selection, particularly in high malaria transmission settings.