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1.
Malar J ; 20(1): 111, 2021 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-33632228

RESUMO

BACKGROUND: Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. The aim of this study was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches. METHODS: High throughput multiplex quantitative real-time PCR method was used to genotype KIR genetic variants and copy number variation and a high-throughput real-time PCR method was developed to genotype HLA-C1 and C2 allotypes for 1344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. RESULTS: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5, and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6 vs 13.2%: p = 0.006, 57.2 vs 66.4%: p = 0.005, 33.2 vs 46.6%: p < 0.001, and 19.7 vs 26.7%: p = 0.014, respectively) or Jinja (7.6 vs 18.1%: p < 0.001, 57.2 vs 63.8%: p = 0.048, 33.2 vs 43.5%: p = 0.002, and 19.7 vs 30.4%: p < 0.001, respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p = 0.043, with no significant difference between Kanungu and Tororo (26.7%), p = 0.296. CONCLUSIONS: The KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes may partly explain differences in transmission intensity of malaria since these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput, multiplex, real-time HLA-C genotyping PCR method developed will be useful in disease-association studies involving large cohorts.

2.
Am J Trop Med Hyg ; 103(1): 485-493, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32372751

RESUMO

Tropical splenomegaly is often associated with malaria and schistosomiasis. In 2014 and 2015, 145 Congolese refugees in western Uganda diagnosed with splenomegaly during predeparture medical examinations underwent enhanced screening for various etiologies. After anecdotal reports of unresolved splenomegaly and complications after U.S. arrival, patients were reassessed to describe long-term clinical progression after arrival in the United States. Post-arrival medical information was obtained through medical chart abstraction in collaboration with state health partners in nine participating states. We evaluated observed splenomegaly duration and associated clinical sequelae between 130 case patients from eastern Congo and 102 controls through adjusted hierarchical Poisson models, accounting for familial clustering. Of the 130 case patients, 95 (73.1%) had detectable splenomegaly after arrival. Of the 85 patients with records beyond 6 months, 45 (52.9%) had persistent splenomegaly, with a median persistence of 14.7 months (range 6.0-27.9 months). Of the 112 patients with available results, 65 (58.0%) patients had evidence of malaria infection, and the mean splenomegaly duration did not differ by Plasmodium species. Refugees with splenomegaly on arrival were 43% more likely to have anemia (adjusted relative risk [aRR]: 1.43, 95% CI: 1.04-1.97). Those with persistent splenomegaly were 60% more likely (adjusted relative risk [aRR]: 1.60, 95% CI: 1.15-2.23) to have a hematologic abnormality, particularly thrombocytopenia (aRR: 5.53, 95% CI: 1.73-17.62), and elevated alkaline phosphatase (aRR: 1.57, 95% CI: 1.03-2.40). Many patients experienced persistent splenomegaly, contradicting literature describing resolution after treatment and removal from an endemic setting. Other possible etiologies should be investigated and effective treatment, beyond treatment for malaria and schistosomiasis, explored.


Assuntos
Anemia/epidemiologia , Eosinofilia/epidemiologia , Malária/epidemiologia , Refugiados , Esquistossomose/epidemiologia , Esplenomegalia/epidemiologia , Trombocitopenia/epidemiologia , Adolescente , Adulto , Fosfatase Alcalina/sangue , Anemia/sangue , Anti-Helmínticos/uso terapêutico , Antimaláricos/uso terapêutico , Combinação Arteméter e Lumefantrina/uso terapêutico , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos de Coortes , República Democrática do Congo/etnologia , Progressão da Doença , Eosinofilia/sangue , Feminino , Hepatite A/epidemiologia , Hepatite B/epidemiologia , Hepatite C/epidemiologia , Humanos , Imunoglobulina M , Lactente , Malária/complicações , Malária/diagnóstico , Malária/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Praziquantel/uso terapêutico , Esquistossomose/complicações , Esquistossomose/tratamento farmacológico , Esplenomegalia/sangue , Esplenomegalia/etiologia , Trombocitopenia/sangue , Estados Unidos/epidemiologia , Adulto Jovem
3.
Malar J ; 19(1): 129, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32228615

RESUMO

BACKGROUND: The World Health Organization (WHO) recommends parasite-based diagnosis of malaria. In recent years, there has been surge in the use of various kinds of nucleic-acid amplification based tests (NAATs) for detection and identification of Plasmodium spp. to support clinical care in high-resource settings and clinical and epidemiological research worldwide. However, these tests are not without challenges, including lack (or limited use) of standards and lack of reproducibility, due in part to variation in protocols amongst laboratories. Therefore, there is a need for rigorous quality control, including a robust external quality assessment (EQA) scheme targeted towards malaria NAATs. To this effect, the WHO Global Malaria Programme worked with the UK National External Quality Assessment Scheme (UK NEQAS) Parasitology and with technical experts to launch a global NAAT EQA scheme in January 2017. METHODS: Panels of NAAT EQA specimens containing five major species of human-infecting Plasmodium at various parasite concentrations and negative samples were created in lyophilized blood (LB) and dried blood spot (DBS) formats. Two distributions per year were sent, containing five LB and five DBS specimens. Samples were tested and validated by six expert referee laboratories prior to distribution. Between 37 and 45 laboratories participated in each distribution and submitted results using the online submission portal of UK NEQAS. Participants were scored based on their laboratory's stated capacity to identify Plasmodium species, and individual laboratory reports were sent which included performance comparison with anonymized peers. RESULTS: Analysis of the first three distributions revealed that the factors that most significantly affected performance were sample format (DBS vs LB), species and parasite density, while laboratory location and the reported methodology used (type of nucleic acid extraction, amplification, or DNA vs RNA target) did not significantly affect performance. Referee laboratories performed better than non-referee laboratories. CONCLUSIONS: Globally, malaria NAAT assays now inform a range of clinical, epidemiological and research investigations. EQA schemes offer a way for laboratories to assess and improve their performance, which is critical to safeguarding the reliability of data and diagnoses especially in situations where various NAAT methodologies and protocols are in use.


Assuntos
Testes Diagnósticos de Rotina/estatística & dados numéricos , Malária/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Plasmodium/isolamento & purificação , Garantia da Qualidade dos Cuidados de Saúde/estatística & dados numéricos , Humanos , Controle de Qualidade , Reprodutibilidade dos Testes , Organização Mundial da Saúde
4.
Malar J ; 19(1): 21, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941490

RESUMO

BACKGROUND: Multiple red blood cell (RBC) variants appear to offer protection against the most severe forms of Plasmodium falciparum malaria. Associations between these variants and uncomplicated malaria are less clear. METHODS: Data from a longitudinal cohort study conducted in 3 sub-counties in Uganda was used to quantify associations between three red blood cell variants Hb [AA, AS, S (rs334)], alpha thalassaemia 3.7 kb deletion, and glucose-6-phosphate dehydrogenase deficiency A-(G6PD 202A genotype) and malaria incidence, parasite prevalence, parasite density (a measure of anti-parasite immunity) and body temperature adjusted for parasite density (a measure of anti-disease immunity). All analyses were adjusted for age, average household entomological inoculation rate, and study site. Results for all variants were compared to those for wild type genotypes. RESULTS: In children, HbAS was associated, compared to wild type, with a lower incidence of malaria (IRR = 0.78, 95% CI 0.66-0.92, p = 0.003), lower parasite density upon infection (PR = 0.66, 95% CI 0.51-0.85, p = 0.001), and lower body temperature for any given parasite density (- 0.13 â„ƒ, 95% CI - 0.21, - 0.05, p = 0.002). In children, HbSS was associated with a lower incidence of malaria (IRR = 0.17, 95% CI 0.04-0.71, p = 0.02) and lower parasite density upon infection (PR = 0.31, 95% CI 0.18-0.54, p < 0.001). α-/αα thalassaemia, was associated with higher parasite prevalence in both children and adults (RR = 1.23, 95% CI 1.06-1.43, p = 0.008 and RR = 1.52, 95% CI 1.04-2.23, p = 0.03, respectively). G6PD deficiency was associated with lower body temperature for any given parasite density only among male hemizygote children (- 0.19 â„ƒ, 95% CI - 0.31, - 0.06, p = 0.003). CONCLUSION: RBC variants were associated with non-severe malaria outcomes. Elucidation of the mechanisms by which they confer protection will improve understanding of genetic protection against malaria.


Assuntos
Eritrócitos/citologia , Malária/sangue , Adulto , Fatores Etários , Distribuição Binomial , Cuidadores , Criança , Pré-Escolar , Estudos de Coortes , Eritrócitos/química , Eritrócitos/classificação , Feminino , Humanos , Incidência , Lactente , Estudos Longitudinais , Malária/epidemiologia , Malária/genética , Malária/parasitologia , Masculino , Parasitemia/sangue , Parasitemia/epidemiologia , Parasitemia/genética , Parasitemia/parasitologia , Prevalência , Estudos Prospectivos , Fatores Sexuais , Uganda/epidemiologia , Adulto Jovem
5.
Nat Ecol Evol ; 3(8): 1253-1264, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31358949

RESUMO

The shift from a hunter-gatherer to an agricultural mode of subsistence is believed to have been associated with profound changes in the burden and diversity of pathogens across human populations. Yet, the extent to which the advent of agriculture affected the evolution of the human immune system remains unknown. Here we present a comparative study of variation in the transcriptional responses of peripheral blood mononuclear cells to bacterial and viral stimuli between Batwa rainforest hunter-gatherers and Bakiga agriculturalists from Uganda. We observed increased divergence between hunter-gatherers and agriculturalists in the early transcriptional response to viruses compared with that for bacterial stimuli. We demonstrate that a significant fraction of these transcriptional differences are under genetic control and we show that positive natural selection has helped to shape population differences in immune regulation. Across the set of genetic variants underlying inter-population immune-response differences, however, the signatures of positive selection were disproportionately observed in the rainforest hunter-gatherers. This result is counter to expectations on the basis of the popularized notion that shifts in pathogen exposure due to the advent of agriculture imposed radically heightened selective pressures in agriculturalist populations.


Assuntos
Leucócitos Mononucleares , Seleção Genética , Agricultura , Humanos , Floresta Úmida , Uganda
6.
Artigo em Inglês | MEDLINE | ID: mdl-30559133

RESUMO

The potential spread of antimalarial drug resistance to Africa, in particular for artemisinins and key partner drugs, is a major concern. We surveyed Plasmodium falciparum genetic markers associated with drug sensitivity on 3 occasions at ∼6-month intervals in 2016 and 2017 at 10 sites representing a range of epidemiological settings in Uganda. For putative drug transporters, we found continued evolution toward wild-type sequences associated with increased sensitivity to chloroquine. For pfcrt K76T, by 2017 the prevalence of the wild type was >60% at all sites and >90% at 6 sites. For the pfmdr1 N86Y and D1246Y alleles, wild type prevalence ranged from 80 to 100%. We found low prevalence of K13 propeller domain mutations, which are associated with artemisinin resistance in Asia, but one mutation previously identified in northern Uganda, 675V, was seen in 2.0% of samples, including 5.5% of those from the 3 northernmost sites. Amplification of the pfmdr1 and plasmepsin2 genes, associated elsewhere with decreased sensitivity to lumefantrine and piperaquine, respectively, was seen in <1% of samples. For the antifolate targets pfdhfr and pfdhps, 5 mutations previously associated with resistance were very common, and the pfdhfr 164L and pfdhps 581G mutations associated with higher-level resistance were seen at multiple sites, although prevalence did not clearly increase over time. Overall, changes were consistent with the selective pressure of the national treatment regimen, artemether-lumefantrine, with increased sensitivity to chloroquine, and with poor efficacy of antifolates. Strong evidence for resistance to artemisinins was not seen. Continued surveillance of markers that predict antimalarial drug sensitivity is warranted.


Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Combinação Arteméter e Lumefantrina/farmacologia , Artemisininas/farmacologia , Ácido Aspártico Endopeptidases/genética , Criança , Cloroquina/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Humanos , Lumefantrina/farmacologia , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas de Protozoários/genética , Quinolinas/farmacologia , Uganda
7.
PLoS One ; 13(9): e0203229, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30222732

RESUMO

BACKGROUND: Evidence for association between sickle cell and alpha thalassemia trait and severe malaria is compelling. However, for these polymorphisms associations with uncomplicated malaria, and for G6PD deficiency associations with uncomplicated and severe malaria, findings have been inconsistent. We studied samples from a three-arm case-control study with the objective of determining associations between common host erythrocyte polymorphisms and both uncomplicated and severe malaria, including different severe malaria phenotypes. METHOD: We assessed hemoglobin abnormalities, α-thalassemia, and G6PD deficiency by molecular methods in 325 children with severe malaria age-matched to 325 children with uncomplicated malaria and 325 healthy community controls. Conditional logistic regression was used to measure associations between specified genotypes and malaria outcomes. RESULTS: No tested polymorphisms offered significant protection against uncomplicated malaria. α-thalassemia homozygotes (_α/_α) had increased risk of uncomplicated malaria (OR 2.40; 95%CI 1.15, 5.03, p = 0.020). HbAS and α-thalassemia heterozygous (_α/αα) genotypes protected against severe malaria compared to uncomplicated malaria (HbAS OR 0.46; 0.23, 0.95, p = 0.036; _α/αα OR 0.51; 0.24, 0.77; p = 0.001) or community (HbAS OR 0.23; 0.11, 0.50; p<0.001; _α/αα; OR 0.49; 0.32, 0.76; p = 0.002) controls. The α-thalassemia homozygous (_α/_α) genotype protected against severe malaria when compared to uncomplicated malaria controls (OR 0.34; 95%CI 0.156, 0.73, p = 0.005), but not community controls (OR 1.03; 0.46, 2.27, p = 0.935). Stratifying by the severe malaria phenotype, compared to community controls, the protective effect of HbAS was limited to children with severe anemia (OR 0.17; 95%CI 0.04, 0.65; p = 0.009) and that of _α/αα to those with altered consciousness (OR 0.24; 0.09, 0.59; p = 0.002). A negative epistatic effect was seen between HbAS and _α/αα; protection compared to uncomplicated malaria controls was not seen in individuals with both polymorphisms (OR 0.45; 0.11, 1.84; p = 0.269). G6PD deficiency was not protective against severe malaria. CONCLUSION: Associations were complex, with HbAS principally protective against severe anemia, _α/αα against altered consciousness, and negative epistasis between the two polymorphisms.


Assuntos
Eritrócitos/metabolismo , Eritrócitos/patologia , Malária/sangue , Malária/complicações , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Genótipo , Deficiência de Glucosefosfato Desidrogenase/sangue , Deficiência de Glucosefosfato Desidrogenase/complicações , Deficiência de Glucosefosfato Desidrogenase/genética , Hemoglobinas Anormais/genética , Humanos , Lactente , Malária/genética , Masculino , Fenótipo , Polimorfismo Genético , Fatores de Risco , Traço Falciforme/sangue , Traço Falciforme/complicações , Traço Falciforme/genética , Uganda , Talassemia alfa/sangue , Talassemia alfa/complicações , Talassemia alfa/genética
8.
Am J Trop Med Hyg ; 97(3): 753-757, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28990911

RESUMO

Contributions of species other than Plasmodium falciparum to human malaria in sub-Saharan Africa are uncertain. We collected blood from children aged 6 months to 10 years diagnosed with malaria by Giemsa-stained blood smears (176 subjects) or histidine rich protein-2-based rapid diagnostic tests (323 subjects) in 2016; 50 samples from each of 10 sites across Uganda were studied to identify infecting species. Of 499 available samples, 474 demonstrated plasmodial infection by polymerase chain reaction amplification of 18S ribosomal RNA genes, including P. falciparum in 472, Plasmodium malariae in 22, Plasmodium ovale in 15, and Plasmodium vivax in four; 435 were pure P. falciparum, two did not contain P. falciparum, and the remainder were mixed infections including P. falciparum. The prevalence of nonfalciparum species varied geographically. Stratifying based on recent history of indoor residual spraying (IRS) of insecticides, nonfalciparum infections were seen in 27/189 (14.8%) samples from sites that received and 13/285 (4.6%) samples from sites that did not receive IRS since 2010 (P = 0.0013). Overall, 39/474 (8.2%) samples from individuals diagnosed with malaria included nonfalciparum infections. Thus, a substantial proportion of episodes of malaria in Uganda include infections with plasmodial species other than P. falciparum.


Assuntos
Malária/epidemiologia , Malária/parasitologia , Plasmodium/classificação , Criança , Pré-Escolar , Humanos , Lactente , Uganda/epidemiologia
9.
Artigo em Inglês | MEDLINE | ID: mdl-28923866

RESUMO

Dihydroartemisinin-piperaquine (DP) has demonstrated excellent efficacy for the treatment and prevention of malaria in Uganda. However, resistance to both components of this regimen has emerged in Southeast Asia. The efficacy of artemether-lumefantrine, the first-line regimen to treat malaria in Uganda, has also been excellent, but continued pressure may select for parasites with decreased sensitivity to lumefantrine. To gain insight into current drug sensitivity patterns, ex vivo sensitivities were assessed and genotypes previously associated with altered drug sensitivity were characterized for 58 isolates collected in Tororo, Uganda, from subjects presenting in 2016 with malaria from the community or as part of a clinical trial comparing DP chemoprevention regimens. Compared to community isolates, those from trial subjects had lower sensitivities to the aminoquinolines chloroquine, monodesethyl amodiaquine, and piperaquine and greater sensitivities to lumefantrine and mefloquine, an observation consistent with DP selection pressure. Compared to results for isolates from 2010 to 2013, the sensitivities of 2016 community isolates to chloroquine, amodiaquine, and piperaquine improved (geometric mean 50% inhibitory concentrations [IC50] = 248, 76.9, and 19.1 nM in 2010 to 2013 and 33.4, 14.9, and 7.5 nM in 2016, respectively [P < 0.001 for all comparisons]), the sensitivity to lumefantrine decreased (IC50 = 3.0 nM in 2010 to 2013 and 5.4 nM in 2016 [P < 0.001]), and the sensitivity to dihydroartemisinin was unchanged (IC50 = 1.4 nM). These changes were accompanied by decreased prevalence of transporter mutations associated with aminoquinoline resistance and low prevalence of polymorphisms recently associated with resistance to artemisinins or piperaquine. Antimalarial drug sensitivities are changing in Uganda, but novel genotypes associated with DP treatment failure in Asia are not prevalent.


Assuntos
Antimaláricos/uso terapêutico , Resistência a Medicamentos/genética , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/genética , Adolescente , Amodiaquina/análogos & derivados , Amodiaquina/uso terapêutico , Artemisininas/uso terapêutico , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Criança , Pré-Escolar , Cloroquina/uso terapêutico , Etanolaminas/uso terapêutico , Feminino , Fluorenos/uso terapêutico , Expressão Gênica , Humanos , Lactente , Concentração Inibidora 50 , Lumefantrina , Malária Falciparum/parasitologia , Masculino , Mefloquina/uso terapêutico , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Testes de Sensibilidade Parasitária , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Quinolinas/uso terapêutico , Uganda , Adulto Jovem
10.
Infect Genet Evol ; 55: 281-287, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28939159

RESUMO

A number of human genetic polymorphisms are prevalent in tropical populations and appear to offer protection against symptomatic and/or severe malaria. We compared the prevalence of four polymorphisms, the sickle hemoglobin mutation (ß globin E6V), the α-thalassemia 3.7kb deletion, glucose-6-phosphate dehydrogenase deficiency caused by the common African variant (G6PD A-), and the CD36 T188G mutation in 1344 individuals residing in districts in eastern (Tororo), south-central (Jinja), and southwestern (Kanungu) Uganda. Genes of interest were amplified, amplicons subjected to mutation-specific restriction endonuclease digestion (for sickle hemoglobin, G6PD A-, and CD36 T188G), reaction products resolved by electrophoresis, and genotypes determined based on the sizes of reaction products. Mutant genotypes were common, with many more heterozygous than homozygous alleles identified. The prevalences (heterozygotes plus homozygotes) of sickle hemoglobin (28% Tororo, 25% Jinja, 7% Kanungu), α-thalassemia (53% Tororo, 45% Jinja, 18% Kanungu) and G6PD A- (29% Tororo, 18% Jinja, 8% Kanungu) were significantly greater in Tororo and Jinja compared to Kanungu (p<0.0001 for all three alleles); prevalences were also significantly greater in Tororo compared to Jinja for α-thalassemia (p=0.03) and G6PD A- (p<0.0001). For the CD36 T188G mutation, the prevalence was significantly greater in Tororo compared to Jinja or Kanungu (27% Tororo, 17% Jinja, 18% Kanungu; p=0.0004 and 0.0017, respectively). Considering ethnicity of study subjects, based on primary language spoken, the prevalence of mutant genotypes was lower in Bantu compared to non-Bantu language speakers, but in the Jinja cohort, the only study population with a marked diversity of language groups, prevalence did not differ between Bantu and non-Bantu speakers. These results indicate marked differences in human genetic features between populations in different regions of Uganda. These differences might be explained by both ethnic variation and by varied malaria risk in different regions of Uganda.


Assuntos
Resistência à Doença/genética , Predisposição Genética para Doença , Malária/epidemiologia , Malária/genética , Polimorfismo Genético , Antígenos CD36/genética , Grupos Étnicos/genética , Feminino , Deficiência de Glucosefosfato Desidrogenase/genética , Humanos , Masculino , Mutação , Prevalência , Deleção de Sequência , Uganda/epidemiologia , alfa-Globinas/genética
11.
Malar J ; 16(1): 125, 2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28327148

RESUMO

BACKGROUND: Plasmodium falciparum genetic polymorphisms that mediate altered drug sensitivity may impact upon virulence. In a cross-sectional study, Ugandan children with infections mutant at pfcrt K76T, pfmdr1 N86Y, or pfmdr1 D1246Y had about one-fourth the odds of symptomatic malaria compared to those with infections with wild type (WT) sequences. However, results may have been confounded by greater likelihood in those with symptomatic disease of higher density mixed infections and/or recent prior treatment that selected for WT alleles. METHODS: Polymorphisms in samples from paired episodes of asymptomatic and symptomatic parasitaemia in 114 subjects aged 4-11 years were followed longitudinally in Tororo District, Uganda. Paired episodes occurred within 3-12 months of each other and had no treatment for malaria in the prior 60 days. The prevalence of WT, mixed, and mutant alleles was determined using multiplex ligase detection reaction-fluorescent microsphere assays. RESULTS: Considering paired episodes in the same subject, the odds of symptomatic malaria were lower for infections with mutant compared to WT or mixed sequence at N86Y (OR 0.26, 95% CI 0.09-0.79, p = 0.018), but not K76T or D1246Y. However, symptomatic episodes (which had higher densities) were more likely than asymptomatic to be mixed (for N86Y OR 2.0, 95% CI 1.04-4.0, p = 0.036). Excluding mixed infections, the odds of symptomatic malaria were lower for infections with mutant compared to WT sequence at N86Y (OR 0.33, 95% CI 0.11-0.98, p = 0.046), but not the other alleles. However, if mixed genotypes were grouped with mutants in this analysis or assuming that mixed infections consisted of 50% WT and 50% mutant genotypes, the odds of symptomatic infection did not differ between infections that were mutant or WT at the studied alleles. CONCLUSIONS: Although infections with only the mutant pfmdr1 86Y genotype were associated with symptomatic infection, this association could primarily be explained by greater parasite densities and therefore greater prevalence of mixed infections in symptomatic children. These results indicate limited association between the tested polymorphisms and risk of symptomatic disease and highlight the value of longitudinal studies for assessing associations between parasite factors and clinical outcomes.


Assuntos
Doenças Assintomáticas , Resistência a Medicamentos , Malária Falciparum/patologia , Parasitemia/patologia , Plasmodium falciparum/classificação , Plasmodium falciparum/efeitos dos fármacos , Polimorfismo Genético , Adulto , Criança , Pré-Escolar , Feminino , Frequência do Gene , Genótipo , Humanos , Lactente , Estudos Longitudinais , Malária Falciparum/parasitologia , Masculino , Mutação de Sentido Incorreto , Parasitemia/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Uganda
12.
J Infect Dis ; 215(4): 631-635, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28039354

RESUMO

We assessed Plasmodium falciparum drug resistance markers in parasites collected in 2012, 2013, and 2015 at 3 sites in Uganda. The prevalence and frequency of parasites with mutations in putative transporters previously associated with resistance to aminoquinolines, but increased sensitivity to lumefantrine (pfcrt 76T; pfmdr1 86Y and 1246Y), decreased markedly at all sites. Antifolate resistance mutations were common, with apparent emergence of mutations (pfdhfr 164L; pfdhps 581G) associated with high-level resistance. K13 mutations linked to artemisinin resistance were uncommon and did not increase over time. Changing malaria treatment practices have been accompanied by profound changes in markers of resistance.


Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos/genética , Genes de Protozoários , Plasmodium falciparum/efeitos dos fármacos , Adolescente , Aminoquinolinas/farmacologia , Artemisininas/farmacologia , Estudos Transversais , DNA de Protozoário/isolamento & purificação , Etanolaminas/farmacologia , Feminino , Fluorenos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Humanos , Lumefantrina , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Plasmodium falciparum/genética , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Análise de Sequência de DNA , Uganda
13.
Antimicrob Agents Chemother ; 59(8): 5061-4, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26033725

RESUMO

We evaluated markers of artemisinin resistance in Plasmodium falciparum isolated in Kampala in 2014. By standard in vitro assays, all isolates were highly sensitive to dihydroartemisinin (DHA). By the ring-stage survival assay, after a 6-h DHA pulse, parasitemia was undetectable in 40 of 43 cultures at 72 h. Two of 53 isolates had nonsynonymous K13-propeller gene polymorphisms but did not have the mutations associated with resistance in Asia. Thus, we did not see evidence for artemisinin resistance in Uganda.


Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Malária Falciparum/tratamento farmacológico , Parasitemia/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Resistência a Medicamentos , Humanos , Malária Falciparum/parasitologia , Dados de Sequência Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas de Protozoários/genética , Uganda
14.
Antimicrob Agents Chemother ; 59(6): 3018-30, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25753626

RESUMO

Changing treatment practices may be selecting for changes in the drug sensitivity of malaria parasites. We characterized ex vivo drug sensitivity and parasite polymorphisms associated with sensitivity in 459 Plasmodium falciparum samples obtained from subjects enrolled in two clinical trials in Tororo, Uganda, from 2010 to 2013. Sensitivities to chloroquine and monodesethylamodiaquine varied widely; sensitivities to quinine, dihydroartemisinin, lumefantrine, and piperaquine were generally good. Associations between ex vivo drug sensitivity and parasite polymorphisms included decreased chloroquine and monodesethylamodiaquine sensitivity and increased lumefantrine and piperaquine sensitivity with pfcrt 76T, as well as increased lumefantrine sensitivity with pfmdr1 86Y, Y184, and 1246Y. Over time, ex vivo sensitivity decreased for lumefantrine and piperaquine and increased for chloroquine, the prevalences of pfcrt K76 and pfmdr1 N86 and D1246 increased, and the prevalences of pfdhfr and pfdhps polymorphisms associated with antifolate resistance were unchanged. In recurrent infections, recent prior treatment with artemether-lumefantrine was associated with decreased ex vivo lumefantrine sensitivity and increased prevalence of pfcrt K76 and pfmdr1 N86, 184F, and D1246. In children assigned chemoprevention with monthly dihydroartemisinin-piperaquine with documented circulating piperaquine, breakthrough infections had increased the prevalence of pfmdr1 86Y and 1246Y compared to untreated controls. The noted impacts of therapy and chemoprevention on parasite polymorphisms remained significant in multivariate analysis correcting for calendar time. Overall, changes in parasite sensitivity were consistent with altered selective pressures due to changing treatment practices in Uganda. These changes may threaten the antimalarial treatment and preventive efficacies of artemether-lumefantrine and dihydroartemisinin-piperaquine, respectively.


Assuntos
Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Amodiaquina/análogos & derivados , Amodiaquina/farmacologia , Antimaláricos , Artemisininas/farmacologia , Pré-Escolar , Cloroquina/farmacologia , Ensaios Clínicos como Assunto , Etanolaminas/farmacologia , Fluorenos/farmacologia , Humanos , Lactente , Lumefantrina , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Testes de Sensibilidade Parasitária , Polimorfismo Genético/genética , Proteínas de Protozoários/genética , Quinina/farmacologia , Quinolinas/farmacologia , Uganda
15.
Am J Trop Med Hyg ; 91(4): 833-843, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25048375

RESUMO

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 - 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36-17.97, P < 0.001 : were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine.


Assuntos
Antimaláricos/uso terapêutico , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium falciparum/genética , Polimorfismo Genético , Proteínas de Protozoários/genética , Substituição de Aminoácidos , Amodiaquina/uso terapêutico , Antimaláricos/farmacologia , Artemeter , Artemisininas/uso terapêutico , Criança , Pré-Escolar , Cloroquina/farmacologia , Conjuntos de Dados como Assunto , Combinação de Medicamentos , Resistência a Medicamentos/genética , Quimioterapia Combinada , Etanolaminas/uso terapêutico , Fluorenos/uso terapêutico , Marcadores Genéticos/genética , Genótipo , Humanos , Lactente , Estimativa de Kaplan-Meier , Lumefantrina , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Fatores de Risco
16.
Am J Trop Med Hyg ; 91(1): 54-61, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24799371

RESUMO

Standard therapy for malaria in Uganda changed from chloroquine to chloroquine + sulfadoxine-pyrimethamine in 2000, and artemether-lumefantrine in 2004, although implementation of each change was slow. Plasmodium falciparum genetic polymorphisms are associated with alterations in drug sensitivity. We followed the prevalence of drug resistance-mediating P. falciparum polymorphisms in 982 samples from Tororo, a region of high transmission intensity, collected from three successive treatment trials conducted during 2003-2012, excluding samples with known recent prior treatment. Considering transporter mutations, prevalence of the mutant pfcrt 76T, pfmdr1 86Y, and pfmdr1 1246Y alleles decreased over time. Considering antifolate mutations, the prevalence of pfdhfr 51I, 59R, and 108N, and pfdhps 437G and 540E were consistently high; pfdhfr 164L and pfdhps 581G were uncommon, but most prevalent during 2008-2010. Our data suggest sequential selective pressures as different treatments were implemented, and they highlight the importance of genetic surveillance as treatment policies change over time.


Assuntos
Resistência a Medicamentos/genética , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Tetra-Hidrofolato Desidrogenase/genética , Antimaláricos/uso terapêutico , Combinação Arteméter e Lumefantrina , Artemisininas/uso terapêutico , Criança , Pré-Escolar , Cloroquina/uso terapêutico , Ensaios Clínicos como Assunto , Combinação de Medicamentos , Resistência a Medicamentos/efeitos dos fármacos , Etanolaminas/uso terapêutico , Feminino , Fluorenos/uso terapêutico , Marcadores Genéticos , Humanos , Lactente , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Malária Falciparum/transmissão , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutação , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Polimorfismo Genético , Proteínas de Protozoários/metabolismo , Pirimetamina/uso terapêutico , Sulfadoxina/uso terapêutico , Tetra-Hidrofolato Desidrogenase/metabolismo , Fatores de Tempo , Uganda/epidemiologia
17.
Malar J ; 13: 102, 2014 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-24636676

RESUMO

BACKGROUND: Reliable measures of anti-malarial resistance are crucial for malaria control. Resistance is typically a complex trait: multiple mutations in a single parasite (a haplotype or genotype) are necessary for elaboration of the resistant phenotype. The frequency of a genetic motif (proportion of parasite clones in the parasite population that carry a given allele, haplotype or genotype) is a useful measure of resistance. In areas of high endemicity, malaria patients generally harbour multiple parasite clones; they have multiplicities of infection (MOIs) greater than one. However, most standard experimental procedures only allow measurement of marker prevalence (proportion of patient blood samples that test positive for a given mutation or combination of mutations), not frequency. It is misleading to compare marker prevalence between sites that have different mean MOIs; frequencies are required instead. METHODS: A Bayesian statistical model was developed to estimate Plasmodium falciparum genetic motif frequencies from prevalence data collected in the field. To assess model performance and computational speed, a detailed simulation study was implemented. Application of the model was tested using datasets from five sites in Uganda. The datasets included prevalence data on markers of resistance to sulphadoxine-pyrimethamine and an average MOI estimate for each study site. RESULTS: The simulation study revealed that the genetic motif frequencies that were estimated using the model were more accurate and precise than conventional estimates based on direct counting. Importantly, the model did not require measurements of the MOI in each patient; it used the average MOI in the patient population. Furthermore, if a dataset included partially genotyped patient blood samples, the model imputed the data that were missing. Using the model and the Ugandan data, genotype frequencies were estimated and four biologically relevant genotypes were identified. CONCLUSIONS: The model allows fast, accurate, reliable estimation of the frequency of genetic motifs associated with resistance to anti-malarials using prevalence data collected from malaria patients. The model does not require per-patient MOI measurements and can easily analyse data from five markers. The model will be a valuable tool for monitoring markers of anti-malarial drug resistance, including markers of resistance to artemisinin derivatives and partner drugs.


Assuntos
Resistência a Medicamentos , Frequência do Gene , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Genótipo , Haplótipos , Humanos , Malária Falciparum/epidemiologia , Modelos Estatísticos , Plasmodium falciparum/classificação , Plasmodium falciparum/efeitos dos fármacos , Prevalência , Uganda
18.
Malar J ; 13: 95, 2014 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-24629020

RESUMO

BACKGROUND: Malaria remains a major public health problem, and its control has been hampered by drug resistance. For a number of drugs, Plasmodium falciparum single nucleotide polymorphisms (SNPs) are associated with altered drug sensitivity and can be used as markers of drug resistance. Several techniques have been studied to assess resistance markers. The most widely used methodology is restriction fragment length polymorphism (RFLP) analysis. The ligase detection reaction fluorescent microsphere (LDR-FM) assay was recently shown to provide high throughput assessment of P. falciparum SNPs associated with drug resistance. The aim of this study was to validate the reliability and accuracy of the LDR-FM assay in a field setting. METHODS: For 223 samples from a clinical trial in Tororo, Uganda in which P. falciparum was identified by blood smear, DNA was extracted from dried blood spots, genes of interest were amplified by PCR, amplicons were analysed by both RFLP and LDR-FM assays, and results were compared. RESULTS: SNP prevalence (wild type/mixed/mutant) with RFLP analysis was 8/5/87% for pfcrt K76T, 34/37/29% for pfmdr1 N86Y, 64/17/19% for pfmdr1 Y184F, and 42/21/37% for pfmdr1 D1246Y. These prevalences with the LDR-FM assay were 7/5/88%, 31/24/45%, 62/20/18%, and 48/19/33% for the four SNPs, respectively. Combining mixed and mutant outcomes for analysis, agreement between the assays was 97% (K=0.77) for pfcrt K76T, 79% (K=0.55) for pfmdr1 N86Y, 83% (K=0.65) for pfmdr1 Y184F, and 91% (K=0.82) for pfmdr1 D1246Y, with most disagreements due to discrepant readings of mixed genotypes. CONCLUSION: The LDR-FM assay provides a high throughput, relatively inexpensive and accurate assay for the surveillance of P. falciparum SNPs associated with drug resistance in resource-limited countries.


Assuntos
Resistência a Medicamentos , Técnicas de Diagnóstico Molecular/métodos , Testes de Sensibilidade Parasitária/métodos , Plasmodium falciparum/genética , Polimorfismo de Nucleotídeo Único , Pré-Escolar , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Lactente , Ligases/metabolismo , Estudos Longitudinais , Malária Falciparum/parasitologia , Masculino , Microesferas , Plasmodium falciparum/efeitos dos fármacos , Reação em Cadeia da Polimerase , Uganda
19.
Stud Fam Plann ; 45(1): 43-58, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24615574

RESUMO

Understanding the transmission dynamics of HIV and other sexually transmitted infections is critically dependent on accurate behavioral data. This study investigates the effect of the mode of questionnaire delivery on the quality of sexual behavior reporting in a 2010 survey conducted in Kampala, Uganda, among 18-24-year-old women. We compare the reported prevalence of five sexual outcomes across three interview modes: traditional face-to-face interviewing (FTFI) in which question rewording was permitted, FTFI administered via computer-assisted personal interviewing (CAPI) in which questions were read as written, and audio computer-assisted self-interviewing (ACASI) in which participants listened to prerecorded questions and entered responses using a computer touchscreen. We then assess the validity of the data by evaluating the reporting of sexual experience against three biological markers. Results suggest that ACASI elicits higher reporting of some key indicators than FTFI does, but self-reports from all interview modes were subject to validity concerns when compared with biomarker data. The study highlights the important role that biomarkers can play in sexual behavior research.


Assuntos
Biomarcadores , Entrevistas como Assunto , Autorrelato , Comportamento Sexual/estatística & dados numéricos , Doenças Sexualmente Transmissíveis/transmissão , Inquéritos e Questionários , Adolescente , Demografia , Feminino , Humanos , Doenças Sexualmente Transmissíveis/epidemiologia , Uganda/epidemiologia , Adulto Jovem
20.
J Infect Dis ; 210(1): 154-7, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24446524

RESUMO

We explored associations between Plasmodium falciparum drug resistance-mediating polymorphisms and clinical presentations in parasitemic children enrolled in a cross-sectional survey in Tororo, Uganda, using a retrospective case-control design. All 243 febrile children (cases) and 243 randomly selected asymptomatic children (controls) were included. In a multivariate analysis adjusting for age, complexity of infection, and parasite density, the prevalence of wild-type genotypes was significantly higher in febrile children compared to asymptomatic children (pfcrt K76T: odds ratio [OR] 4.41 [95% confidence interval {CI}, 1.28-15.1]; pfmdr1 N86Y: OR 4.08 [95% CI, 2.01-8.31], and pfmdr1 D1246Y: OR 4.90 [95% CI, 1.52-15.8]), suggesting greater virulence for wild-type parasites.


Assuntos
Resistência a Medicamentos , Malária Falciparum/patologia , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Polimorfismo Genético , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos Transversais , Feminino , Humanos , Lactente , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmodium falciparum/isolamento & purificação , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/genética , Estudos Retrospectivos , Uganda , Virulência
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