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1.
Nat Commun ; 12(1): 909, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568678

RESUMO

Malaria control may be enhanced by targeting reservoirs of Plasmodium falciparum transmission. One putative reservoir is asymptomatic malaria infections and the scale of their contribution to transmission in natural settings is not known. We assess the contribution of asymptomatic malaria to onward transmission using a 14-month longitudinal cohort of 239 participants in a high transmission site in Western Kenya. We identify P. falciparum in asymptomatically- and symptomatically-infected participants and naturally-fed mosquitoes from their households, genotype all parasites using deep sequencing of the parasite genes pfama1 and pfcsp, and use haplotypes to infer participant-to-mosquito transmission through a probabilistic model. In 1,242 infections (1,039 in people and 203 in mosquitoes), we observe 229 (pfcsp) and 348 (pfama1) unique parasite haplotypes. Using these to link human and mosquito infections, compared with symptomatic infections, asymptomatic infections more than double the odds of transmission to a mosquito among people with both infection types (Odds Ratio: 2.56; 95% Confidence Interval (CI): 1.36-4.81) and among all participants (OR 2.66; 95% CI: 2.05-3.47). Overall, 94.6% (95% CI: 93.1-95.8%) of mosquito infections likely resulted from asymptomatic infections. In high transmission areas, asymptomatic infections are the major contributor to mosquito infections and may be targeted as a component of transmission reduction.


Assuntos
Anopheles/parasitologia , Malária Falciparum/parasitologia , Malária Falciparum/transmissão , Mosquitos Vetores/parasitologia , Plasmodium falciparum/genética , Adulto , Animais , Anopheles/fisiologia , Infecções Assintomáticas/epidemiologia , Estudos de Coortes , Feminino , Genótipo , Humanos , Quênia/epidemiologia , Estudos Longitudinais , Malária Falciparum/epidemiologia , Masculino , Mosquitos Vetores/fisiologia , Plasmodium falciparum/classificação , Plasmodium falciparum/isolamento & purificação
2.
BMC Infect Dis ; 21(1): 44, 2021 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-33422001

RESUMO

BACKGROUND: Transmission stemming from asymptomatic infections is increasingly being recognized as a threat to malaria elimination. In many regions, malaria transmission is seasonal. It is not well understood whether Plasmodium falciparum modulates its investment in transmission to coincide with seasonal vector abundance. METHODS: We sampled 1116 asymptomatic individuals in the wet season, when vectors are abundant, and 1743 in the dry season, in two sites in western Kenya, representing different transmission intensities (Chulaimbo, moderate transmission, and Homa Bay, low transmission). Blood samples were screened for P. falciparum by qPCR, and gametocytes by pfs25 RT-qPCR. RESULTS: Parasite prevalence by qPCR was 27.1% (Chulaimbo, dry), 48.2% (Chulaimbo, wet), 9.4% (Homabay, dry), and 7.8% (Homabay, wet). Mean parasite densities did not differ between seasons (P = 0.562). pfs25 transcripts were detected in 119/456 (26.1%) of infections. In the wet season, fewer infections harbored detectable gametocytes (22.3% vs. 33.8%, P = 0.009), but densities were 3-fold higher (wet: 3.46 transcripts/uL, dry: 1.05 transcripts/uL, P < 0.001). In the dry season, 4.0% of infections carried gametocytes at moderate-to-high densities likely infective (> 1 gametocyte per 2 uL blood), compared to 7.9% in the wet season. Children aged 5-15 years harbored 76.7% of infections with gametocytes at moderate-to-high densities. CONCLUSIONS: Parasites increase their investment in transmission in the wet season, reflected by higher gametocyte densities. Despite increased gametocyte densities, parasite density remained similar across seasons and were often below the limit of detection of microscopy or rapid diagnostic test, thus a large proportion of infective infections would escape population screening in the wet season. Seasonal changes of gametocytemia in asymptomatic infections need to be considered when designing malaria control measures.


Assuntos
Portador Sadio/parasitologia , Malária Falciparum/parasitologia , Plasmodium falciparum/fisiologia , Adolescente , Infecções Assintomáticas/epidemiologia , Portador Sadio/epidemiologia , Criança , Pré-Escolar , Feminino , Humanos , Quênia/epidemiologia , Malária Falciparum/epidemiologia , Masculino , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/isolamento & purificação , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , Estações do Ano
3.
Anal Chem ; 93(4): 2097-2105, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33464825

RESUMO

In many countries targeting malaria elimination, persistent malaria infections can have parasite loads significantly below the lower limit of detection (LLOD) of standard diagnostic techniques, making them difficult to identify and treat. The most sensitive diagnostic methods involve amplification and detection of Plasmodium DNA by polymerase chain reaction (PCR), which requires expensive thermal cycling equipment and is difficult to deploy in resource-limited settings. Isothermal DNA amplification assays have been developed, but they require complex primer design, resulting in high nonspecific amplification, and show a decrease in sensitivity than PCR methods. Here, we have used a computational approach to design a novel isothermal amplification assay with a simple primer design to amplify P. falciparum DNA with analytical sensitivity comparable to PCR. We have identified short DNA sequences repeated throughout the parasite genome to be used as primers for DNA amplification and demonstrated that these primers can be used, without modification, to isothermally amplify P. falciparum parasite DNA via strand displacement amplification. Our novel assay shows a LLOD of ∼1 parasite/µL within a 30 min amplification time. The assay was demonstrated with clinical samples using patient blood and saliva. We further characterized the assay using direct amplicon next-generation sequencing and modified the assay to work with a visual readout. The technique developed here achieves similar analytical sensitivity to current gold standard PCR assays requiring a fraction of time and resources for PCR. This highly sensitive isothermal assay can be more easily adapted to field settings, making it a potentially useful tool for malaria elimination.


Assuntos
DNA de Protozoário/genética , Malária Falciparum/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Plasmodium falciparum/genética , Sequências Repetitivas de Ácido Nucleico/genética , DNA de Protozoário/isolamento & purificação , Humanos , Limite de Detecção , Plasmodium falciparum/isolamento & purificação , Reprodutibilidade dos Testes
4.
BMC Infect Dis ; 21(1): 91, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33478414

RESUMO

BACKGROUND: Informed decision making is underlined by all tiers in the health system. Poor data record system coupled with under- (over)-reporting of malaria cases affects the country's malaria elimination activities. Thus, malaria data at health facilities and health offices are important particularly to monitor and evaluate the elimination progresses. This study was intended to assess overall reported malaria cases, reporting quality, spatiotemporal trends and factors associated in Gedeo zone, South Ethiopia. METHODS: Past 8 years retrospective data stored in 17 health centers and 5 district health offices in Gedeo Zone, South Ethiopia were extracted. Malaria cases data at each health center with sociodemographic information, between January 2012 and December 2019, were included. Meteorological data were obtained from the national meteorology agency of Ethiopia. The data were analyzed using Stata 13. RESULTS: A total of 485,414 suspected cases were examined for malaria during the previous 8 years at health centers. Of these suspects, 57,228 (11.79%) were confirmed malaria cases with an overall decline during the 8-year period. We noted that 3758 suspected cases and 467 confirmed malaria cases were not captured at the health offices. Based on the health centers records, the proportions of Plasmodium falciparum (49.74%) and P. vivax (47.59%) infection were nearly equivalent (p = 0.795). The former was higher at low altitudes while the latter was higher at higher altitudes. The over 15 years of age group accounted for 11.47% of confirmed malaria cases (p < 0.001). There was high spatiotemporal variation: the highest case record was during Belg (12.52%) and in Dilla town (18,150, 13.17%, p < 0.001) which is located at low altitude. Monthly rainfall and minimum temperature exhibited strong associations with confirmed malaria cases. CONCLUSION: A notable overall decline in malaria cases was observed during the eight-year period. Both P. falciparum and P. vivax were found at equivalent endemicity level; hence control measures should continue targeting both species. The noticed under reporting, the high malaria burden in urban settings, low altitudes and Belg season need spatiotemporal consideration by the elimination program.


Assuntos
Malária/epidemiologia , Altitude , Demografia , Notificação de Doenças/normas , Notificação de Doenças/estatística & dados numéricos , Etiópia/epidemiologia , Feminino , Humanos , Malária/diagnóstico , Malária/parasitologia , Malária/prevenção & controle , Masculino , Meteorologia , Análise Multivariada , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/isolamento & purificação , Prevalência , Estudos Retrospectivos , Estações do Ano
5.
PLoS One ; 15(12): e0244457, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382787

RESUMO

BACKGROUND: Plasmodium falciparum histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) are exclusively recommended for malaria diagnosis in Uganda; however, their functionality can be affected by parasite-related factors that have not been investigated in field settings. METHODS: Using a cross-sectional design, we analysed 219 RDT-/microscopy+ and 140 RDT+/microscopy+ dried blood spots obtained from symptomatic children aged 2-10 years from 48 districts in Uganda between 2017 and 2019. We aimed to investigate parasite-related factors contributing to false RDT results by molecular characterization of parasite isolates. ArcGIS software was used to map the geographical distribution of parasites. Statistical analysis was performed using chi-square or Fisher's exact tests, with P ≤ 0.05 indicating significance. Odds ratios (ORs) were used to assess associations, while logistic regression was performed to explore possible factors associated with false RDT results. RESULTS: The presence of parasite DNA was confirmed in 92.5% (332/359) of the blood samples. The levels of agreement between the HRP2 RDT and PCR assay results in the (RDT+/microscopy+) and (RDT-/microscopy+) sample subsets were 97.8% (137/140) and 10.9% (24/219), respectively. Factors associated with false-negative RDT results in the (RDT-/microscopy+) samples were parasite density (<1,000/µl), pfhrp2/3 gene deletion and non-P. falciparum species (aOR 2.65, 95% CI: 1.62-4.38, P = 0.001; aOR 4.4, 95% CI 1.72-13.66, P = 0.004; and aOR 18.65, 95% CI: 5.3-38.7, P = 0.001, respectively). Overall, gene deletion and non-P. falciparum species contributed to 12.3% (24/195) and 19.0% (37/195) of false-negative RDT results, respectively. Of the false-negative RDTs results, 80.0% (156/195) were from subjects with low-density infections (< 25 parasites per 200 WBCs or <1,000/µl). CONCLUSION: This is the first evaluation and report of the contributions of pfhrp2/3 gene deletion, non-P. falciparum species, and low-density infections to false-negative RDT results under field conditions in Uganda. In view of these findings, the use of HRP2 RDTs should be reconsidered; possibly, switching to combination RDTs that target alternative antigens, particularly in affected areas, may be beneficial. Future evaluations should consider larger and more representative surveys covering other regions of Uganda.


Assuntos
Antígenos de Protozoários/isolamento & purificação , Malária Falciparum/diagnóstico , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários/isolamento & purificação , Kit de Reagentes para Diagnóstico/estatística & dados numéricos , Antígenos de Protozoários/imunologia , Criança , Pré-Escolar , Estudos Transversais , DNA de Protozoário/isolamento & purificação , Teste em Amostras de Sangue Seco/instrumentação , Teste em Amostras de Sangue Seco/estatística & dados numéricos , Monitoramento Epidemiológico , Reações Falso-Negativas , Reações Falso-Positivas , Feminino , Humanos , Malária Falciparum/epidemiologia , Malária Falciparum/parasitologia , Malária Falciparum/transmissão , Masculino , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Reação em Cadeia da Polimerase/estatística & dados numéricos , Prevalência , Proteínas de Protozoários/imunologia , Uganda/epidemiologia
6.
BMC Infect Dis ; 20(1): 825, 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33176716

RESUMO

BACKGROUND: Light microscopy is often used for malaria diagnosis in the field. However, it is time-consuming and quality of the results depends heavily on the skill of microscopists. Automating malaria light microscopy is a promising solution, but it still remains a challenge and an active area of research. Current tools are often expensive and involve sophisticated hardware components, which makes it hard to deploy them in resource-limited areas. RESULTS: We designed an Android mobile application called Malaria Screener, which makes smartphones an affordable yet effective solution for automated malaria light microscopy. The mobile app utilizes high-resolution cameras and computing power of modern smartphones to screen both thin and thick blood smear images for P. falciparum parasites. Malaria Screener combines image acquisition, smear image analysis, and result visualization in its slide screening process, and is equipped with a database to provide easy access to the acquired data. CONCLUSION: Malaria Screener makes the screening process faster, more consistent, and less dependent on human expertise. The app is modular, allowing other research groups to integrate their methods and models for image processing and machine learning, while acquiring and analyzing their data.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Malária Falciparum/diagnóstico por imagem , Programas de Rastreamento/métodos , Microscopia/métodos , Plasmodium falciparum/isolamento & purificação , Smartphone , Confiabilidade dos Dados , Humanos , Aprendizado de Máquina , Malária Falciparum/parasitologia , Sensibilidade e Especificidade , Software
8.
Front Immunol ; 11: 1870, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32983106

RESUMO

Coronavirus disease 2019 (COVID-19) which is caused by the novel SARS-CoV-2 virus is a severe flu-like illness which is associated with hyperinflammation and immune dysfunction. The virus induces a strong T and B cell response but little is known about the immune pathology of this viral infection. Acute Plasmodium falciparum malaria also causes acute clinical illness and is characterized by hyperinflammation due to the strong production of pro-inflammatory cytokines and a massive activation of T cells. In malaria, T cells express a variety of co-inhibitory receptors which might be a consequence of their activation but also might limit their overwhelming function. Thus, T cells are implicated in protection as well as in pathology. The outcome of malaria is thought to be a consequence of the balance between co-activation and co-inhibition of T cells. Following the hypothesis that T cells in COVID-19 might have a similar, dual function, we comprehensively characterized the differentiation (CCR7, CD45RO) and activation status (HLA-DR, CD38, CD69, CD226), the co-expression of co-inhibitory molecules (PD1, TIM-3, LAG-3, BTLA, TIGIT), as well as the expression pattern of the transcription factors T-bet and eomes of CD8+ and CD4+ T cells of PBMC of n = 20 SARS-CoV-2 patients compared to n = 10 P. falciparum infected patients and n = 13 healthy controls. Overall, acute COVID-19 and malaria infection resulted in a comparably elevated activation and altered differentiation status of the CD8+ and CD4+ T cell populations. T effector cells of COVID-19 and malaria patients showed higher frequencies of the inhibitory receptors T-cell immunoglobulin mucin-3 (TIM-3) and Lymphocyte-activation gene-3 (LAG-3) which was linked to increased activation levels and an upregulation of the transcription factors T-bet and eomes. COVID-19 patients with a more severe disease course showed higher levels of LAG-3 and TIM-3 than patients with a mild disease course. During recovery, a rapid normalization of these inhibitory receptors could be observed. In summary, comparing the expression of different co-inhibitory molecules in CD8+ and CD4+ T cells in COVID-19 vs. malaria, there is a transient increase of the expression of certain inhibitory receptors like LAG-3 and TIM-3 in COVID-19 in the overall context of acute immune activation.


Assuntos
Antígenos CD/metabolismo , Betacoronavirus/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Coronavirus/imunologia , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Ativação Linfocitária/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/isolamento & purificação , Pneumonia Viral/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Doença Aguda , Adulto , Idoso , Células Cultivadas , Estudos de Coortes , Infecções por Coronavirus/virologia , Feminino , Humanos , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/virologia , Receptor de Morte Celular Programada 1/metabolismo , Índice de Gravidade de Doença
9.
PLoS One ; 15(8): e0237786, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822376

RESUMO

Plasmodium falciparum malaria killed 451,000 people in 2017. Merozoites, the stage of the parasite that invades RBCs, are a logical target for vaccine development. Treatment with the protease inhibitor E64 followed by filtration through a 1.2 µm filter is being used to purify merozoites for immunologic assays. However, there have been no studies to determine the effect of these treatments on the susceptibility of merozoites to complement or antibodies. To address this gap, we purified merozoites with or without E64 followed by filtration through either a 1.2 or 2.7 µm filter, or no filtration. Merozoites were then incubated in either 10% fresh or heat-inactivated serum followed by surface staining and flow cytometry with monoclonal antibodies against the complement effector molecules C3b or C5b9. To determine the effect of anti-merozoite antibodies, we incubated merozoites with MAb5.2, a mouse monoclonal antibody that targets the merozoite surface protein 1. We used an amine-reactive fluorescent dye to measure membrane integrity. Treatment with E64 resulted in an insignificant increase in the proportion of merozoites that were C3b positive but in a significant increase in the proportion that were C5b9 positive. Filtration increased the proportion of merozoites that were either C3b or C5b9-positive. The combination of filtration and E64 treatment resulted in marked deposition of C3b and C5b9. MAb5.2 induced greater complement deposition than serum alone or an IgG2b isotype control. The combination of E64 treatment, filtration, and MAb5.2 resulted in very rapid and significant deposition of C5b9. Filtration through the 1.2 µm filter selected a population of merozoites with greater membrane integrity, but their integrity deteriorated rapidly upon exposure to serum. We conclude that E64 treatment and filtration increase the susceptibility of merozoites to complement and antibody. Filtered or E64-treated merozoites are not suitable for immunologic studies that address the efficacy of antibodies in vitro.


Assuntos
Merozoítos/efeitos dos fármacos , Merozoítos/isolamento & purificação , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/isolamento & purificação , Inibidores de Proteases/farmacologia , Animais , Anticorpos Antiprotozoários/imunologia , Ativação do Complemento/efeitos dos fármacos , Filtração , Citometria de Fluxo , Humanos , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Merozoítos/imunologia , Camundongos , Plasmodium falciparum/imunologia
10.
PLoS One ; 15(8): e0236920, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745085

RESUMO

BACKGROUND: Twenty-seven villages were selected in southwest Burkina Faso to implement new vector control strategies in addition to long lasting insecticidal nets (LLINs) through a Randomized Controlled Trial (RCT). We conducted entomological surveys in the villages during the dry cold season (January 2017), dry hot season (March 2017) and rainy season (June 2017) to describe malaria vectors bionomics, insecticide resistance and transmission prior to this trial. METHODS: We carried out hourly catches (from 17:00 to 09:00) inside and outside 4 houses in each village using the Human Landing Catch technique. Mosquitoes were identified using morphological taxonomic keys. Specimens belonging to the Anopheles gambiae complex and Anopheles funestus group were identified using molecular techniques as well as detection of Plasmodium falciparum infection and insecticide resistance target-site mutations. RESULTS: Eight Anopheles species were detected in the area. Anopheles funestus s.s was the main vector during the dry cold season. It was replaced by Anopheles coluzzii during the dry hot season whereas An. coluzzii and An. gambiae s.s. were the dominant species during the rainy season. Species composition of the Anopheles population varied significantly among seasons. All insecticide resistance mechanisms (kdr-w, kdr-e and ace-1 target site mutations) investigated were found in each members of the An. gambiae complex but at different frequencies. We observed early and late biting phenotypes in the main malaria vector species. Entomological inoculation rates were 2.61, 2.67 and 11.25 infected bites per human per month during dry cold season, dry hot season and rainy season, respectively. CONCLUSION: The entomological indicators of malaria transmission were high despite the universal coverage with LLINs. We detected early and late biting phenotypes in the main malaria vector species as well as physiological insecticide resistance mechanisms. These data will be used to evaluate the impact of complementary tools to LLINs in an upcoming RCT.


Assuntos
Anopheles , Resistência a Inseticidas/genética , Malária Falciparum/transmissão , Mosquitos Vetores/genética , Animais , Anopheles/classificação , Anopheles/genética , Anopheles/parasitologia , Burkina Faso/epidemiologia , Culex/classificação , Culex/genética , Culex/parasitologia , Culicidae/classificação , Culicidae/genética , Culicidae/parasitologia , Ecologia , Genótipo , Humanos , Malária Falciparum/prevenção & controle , Controle de Mosquitos/métodos , Controle de Mosquitos/organização & administração , Mosquitos Vetores/classificação , Mosquitos Vetores/parasitologia , Plasmodium falciparum/isolamento & purificação , Estações do Ano
11.
Am J Trop Med Hyg ; 103(5): 1902-1909, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32840197

RESUMO

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


Assuntos
Antígenos de Protozoários/imunologia , Testes Diagnósticos de Rotina/métodos , L-Lactato Desidrogenase/imunologia , Malária Falciparum/diagnóstico , Plasmodium falciparum/isolamento & purificação , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Antígenos de Protozoários/genética , Criança , Pré-Escolar , Estudos Transversais , Demografia , Etiópia , Feminino , Geografia , Humanos , L-Lactato Desidrogenase/genética , Malária Falciparum/parasitologia , Masculino , Microscopia , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , Sensibilidade e Especificidade , Adulto Jovem
12.
Exp Parasitol ; 218: 107969, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32858043

RESUMO

Invasion of human red blood cells (RBCs) by Plasmodium parasites is a crucial yet poorly characterised phenotype. Two-color flow cytometry (2cFCM) promises to be a very sensitive and high throughput method for phenotyping parasite invasion. However, current protocols require high (~1.0%) parasitemia for assay set-up and need to be adapted for low parasitemia samples, which are becoming increasingly common in low transmission settings. Background fluorescence from nuclei-containing uninfected RBCs and high autologous reinvasion rates (merozoite invasion of donor uninfected RBCs present at 50% assay volume) are some of the limitations to the method's sensitivity to enumerate low parasitemia (<0.5%) with nucleic acid-based stains. Here, we describe modifications for plating unlabeled donor to labeled target RBCs per assay well and for gating parasitemia, that produces accurate quantifications of low reinvasion parasitemia. Plasmodium falciparum 3D7, Dd2 and field isolates at various low and high parasitemia (0.05%-2.0%) were used to set-up SyBr Green 1-based 2cFCM invasion assays. Target RBCs were labeled with CTFR proliferation dye. We show that this dye combination allowed for efficient parasite invasion into target RBCs and that a 1:3 ratio of unlabeled to labeled RBCs per assay greatly skewed autologous reinvasion (p < 0.001). Accuracy of quantifying reinvasion was limited to an assay parasitemia of 0.02% with minimal background interference. Invasion inhibition by enzymatic treatments increased averagely by 10% (p<0.05) across the entire parasitemia range. The effect was greater for samples with <0.5% parasitemia. Overall, a more sensitive method for phenotyping invasion of low P. falciparum parasitemia is described.


Assuntos
Citometria de Fluxo/métodos , Malária Falciparum/parasitologia , Parasitemia/parasitologia , Plasmodium falciparum/isolamento & purificação , Rastreamento de Células/métodos , Corantes , Eritrócitos/parasitologia , Humanos , Fenótipo , Plasmodium falciparum/classificação , Plasmodium falciparum/fisiologia , Recidiva , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos
13.
BMC Infect Dis ; 20(1): 513, 2020 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-32677899

RESUMO

BACKGROUND: Imported falciparum malaria from Africa has become a key public health challenge in Guizhou Province since 2012. Understanding the polymorphisms of molecular markers of drug resistance can guide selection of antimalarial drugs for the treatment of malaria. This study was aimed to analyze the polymorphisms of pfcrt, pfmdr1, and K13-propeller among imported falciparum malaria cases in Guizhou Province, China. METHOD: Fifty-five imported falciparum malaria cases in Guizhou Province during 2012-2016 were included in this study. Their demographic information and filter paper blood samples were collected. Genomic DNA of Plasmodium falciparum was extracted from the blood samples, and polymorphisms of pfcrt, pfmdr1, and K13-propeller were analyzed with nested PCR amplification followed by sequencing. Data were analyzed with the SPSS17.0 software. RESULTS: The prevalence of pfcrt K76T, pfmdr1 N86Y, and pfmdr1 Y184F mutation was 56.6, 22.2, and 72.2%, respectively, in imported falciparum malaria cases in Guizhou Province. We detected two mutant haplotypes of pfcrt, IET and MNT, with IET being more commonly found (54.7%), and five mutant haplotypes of pfmdr1, of which NFD was the most frequent (53.7%). There were totally 10 combined haplotypes of pfcrt and pfmdr1, of which the haplotype IETNFD possessed a predominance of 28.8%. In addition, three nonsynonymous mutations (S459T, C469F, and V692L) and two synonymous mutations (R471R and V589V) were detected in K13-propeller, all having prevalence less than 6.0%. In particular, a candidate K13 resistance mutation, C469F, was identified for the first time from Democratic Republic of the Congo with the prevalence of 2.0%. CONCLUSIONS: The high prevalence of IET haplotype of pfcrt and NFD haplotype of pfmdr1 suggests the presence of chloroquine, artemether/lumefantrine, and dihydroartemisinin/piperaquine resistance in these cases. Therefore cautions should be made to artemisinin therapy for P. falciparum in Africa. Continuous monitoring of anti-malarial drug efficacy in imported malaria cases is helpful for optimizing antimalarial drug therapy in Guizhou Province, China.


Assuntos
Doenças Transmissíveis Importadas/parasitologia , Resistência a Medicamentos/genética , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Polimorfismo Genético , Proteínas de Protozoários/genética , Adulto , África/epidemiologia , Substituição de Aminoácidos/genética , Antimaláricos/uso terapêutico , China/epidemiologia , Estudos de Coortes , Feminino , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Masculino , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Plasmodium falciparum/isolamento & purificação , Doença Relacionada a Viagens
14.
BMC Infect Dis ; 20(1): 487, 2020 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-32646433

RESUMO

BACKGROUND: Genetic diversity of ABO blood, glucose-6-phosphate dehydrogenase (G6PD) deficiency and haemoglobin type and their ability to protect against malaria vary geographically, ethnically and racially. No study has been carried out in populations resident in malaria regions in western Kenya. METHOD: A total of 574 malaria cases (severe malaria anaemia, SMA = 137 and non-SMA = 437) seeking treatment at Vihiga County and Referral Hospital in western Kenya, were enrolled and screened for ABO blood group, G6PD deficiency and haemoglobin genotyped in a hospital-based cross-sectional study. RESULT: When compared to blood group O, blood groups A, AB and B were not associated with SMA (P = 0.380, P = 0.183 and P = 0.464, respectively). Further regression analysis revealed that the carriage of the intermediate status of G6PD was associated with risk to SMA (OR = 1.52, 95%CI = 1.029-2.266, P = 0.035). There was, however, no association between AS and SS with severe malaria anaemia. Co-occurrence of both haemoglobin type and G6PD i.e. the AA/intermediate was associated with risk to SMA (OR = 1.536, 95%CI = 1.007-2.343, P = 0.046) while the carriage of the AS/normal G6PD was associated with protection against SMA (OR = 0.337, 95%CI = 0.156-0.915, P = 0.031). CONCLUSION: Results demonstrate that blood group genotypes do not have influence on malaria disease outcome in this region. Children in Vihiga with blood group O have some protection against malaria. However, the intermediate status of G6PD is associated with risk of SMA. Further, co-inheritance of sickle cell and G6PD status are important predictors of malaria disease outcome. This implies combinatorial gene function in influencing disease outcome.


Assuntos
Sistema ABO de Grupos Sanguíneos/genética , Genótipo , Deficiência de Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/sangue , Hemoglobinas/genética , Malária Falciparum/sangue , Malária Falciparum/epidemiologia , Pré-Escolar , Estudos Transversais , Feminino , Humanos , Lactente , Quênia/epidemiologia , Malária Falciparum/parasitologia , Masculino , Fenótipo , Plasmodium falciparum/isolamento & purificação , Polimorfismo Genético , Risco , Traço Falciforme/genética
15.
Am J Trop Med Hyg ; 103(2): 778-784, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32602431

RESUMO

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


Assuntos
Antígenos de Protozoários/imunologia , Malária Falciparum/diagnóstico , Parasitemia/diagnóstico , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Adolescente , Adulto , Infecções Assintomáticas , Criança , Pré-Escolar , Reações Falso-Positivas , Feminino , Humanos , Lactente , Masculino , Microscopia , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , RNA Ribossômico 18S/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Fatores de Tempo , Uganda
16.
BMC Infect Dis ; 20(1): 533, 2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32698879

RESUMO

BACKGROUND: Plasmodium falciparum parasites, which could harbour anti-malaria drug resistance genes, are commonly detected in blood donors in malaria-endemic areas. Notwithstanding, anti-malaria drug resistant biomarkers have not been characterized in blood donors with asymptomatic P. falciparum infection. METHODS: A total of 771 blood donors were selected from five districts in the Greater Accra Region, Ghana. Each donor sample was screened with malaria rapid diagnostic test (RDT) kit and parasitaemia quantified microscopically. Dried blood spots from malaria positive samples were genotyped for P. falciparum chloroquine resistance transporter (Pfcrt), P. falciparum multi-drug resistance (Pfmdr1), P. falciparum dihydropteroate-synthetase (Pfdhps), P. falciparum dihydrofolate-reductase (Pfdhfr) and Kelch 13 propeller domain on chromosome 13 (Kelch 13) genes. RESULTS: Of the 771 blood donors, 91 (11.8%) were positive by RDT. Analysis of sequence reads indicated successful genotyping of Pfcrt, Pfmdr1, Pfdhfr, Pfdhps and Kelch 13 genes in 84.6, 81.3, 86.8, 86.9 and 92.3% of the isolates respectively. Overall, 21 different mutant haplotypes were identified in 69 isolates (75.8%). In Pfcrt, CVIET haplotype was observed in 11.6% samples while in Pfmdr1, triple mutation (resulting in YFN haplotype) was detected in 8.1% of isolates. In Pfdhfr gene, triple mutation resulting in IRNI haplotype and in Pfdhps gene, quintuple mutation resulting in AGESS haplotype was identified in 17.7% parasite isolates. Finally, five non-synonymous Kelch 13 alleles were detected; C580Y (3.6%), P615L (4.8%), A578S (4.8%), I543V (2.4%) and A676S (1.2%) were detected. CONCLUSION: Results obtained in this study indicated various frequencies of mutant alleles in Pfcrt, Pfmdr1, Pfdhfr, Pfdhps and Kelch 13 genes from P. falciparum infected blood donors. These alleles could reduce the efficacy of standard malaria treatment in transfusion-transmitted malaria cases. Incorporating malaria screening into donor screening protocol to defer infected donors is therefore recommended.


Assuntos
Doadores de Sangue , Resistência Microbiana a Medicamentos/genética , Resistência a Múltiplos Medicamentos/genética , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Plasmodium falciparum/genética , Adolescente , Adulto , Alelos , Antimaláricos/uso terapêutico , Doenças Assintomáticas , Biomarcadores , Cloroquina/uso terapêutico , Estudos Transversais , Di-Hidropteroato Sintase/genética , Feminino , Frequência do Gene , Gana/epidemiologia , Haplótipos , Humanos , Repetição Kelch/genética , Malária Falciparum/diagnóstico , Malária Falciparum/parasitologia , Masculino , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Plasmodium falciparum/isolamento & purificação , Prevalência , Proteínas de Protozoários/genética , Tetra-Hidrofolato Desidrogenase/genética , Adulto Jovem
17.
BMC Infect Dis ; 20(1): 503, 2020 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-32660434

RESUMO

BACKGROUND: Understanding the relationship between malaria infection risk and disease outcomes represents a fundamental component of morbidity and mortality burden estimations. Contemporary data on severe malaria risks among populations of different parasite exposures are scarce. Using surveillance data, we compared rates of paediatric malaria hospitalisation in areas of varying parasite exposure levels. METHODS: Surveillance data at five public hospitals; Jinja, Mubende, Kabale, Tororo, and Apac were assembled among admissions aged 1 month to 14 years between 2017 and 2018. The address of each admission was used to define a local catchment population where national census data was used to define person-year-exposure to risk. Within each catchment, historical infection prevalence was assembled from previously published data and current infection prevalence defined using 33 population-based school surveys among 3400 children. Poisson regression was used to compute the overall and site-specific incidences with 95% confidence intervals. RESULTS: Both current and historical Plasmodium falciparum prevalence varied across the five sites. Current prevalence ranged from < 1% in Kabale to 54% in Apac. Overall, the malaria admission incidence rate (IR) was 7.3 per 1000 person years among children aged 1 month to 14 years of age (95% CI: 7.0, 7.7). The lowest rate was described at Kabale (IR = 0.3; 95 CI: 0.1, 0.6) and highest at Apac (IR = 20.3; 95 CI: 18.9, 21.8). There was a correlation between IR across the five sites and the current parasite prevalence in school children, though findings were not statistically significant. Across all sites, except Kabale, malaria admissions were concentrated among young children, 74% were under 5 years. The median age of malaria admissions at Kabale hospital was 40 months (IQR 20, 72), and at Apac hospital was 36 months (IQR 18, 69). Overall, severe anaemia (7.6%) was the most common presentation and unconsciousness (1.8%) the least common. CONCLUSION: Malaria hospitalisation rates remain high in Uganda particularly among young children. The incidence of hospitalized malaria in different locations in Uganda appears to be influenced by past parasite exposure, immune acquisition, and current risks of infection. Interruption of transmission through vector control could influence age-specific severe malaria risk.


Assuntos
Anemia/etiologia , Hospitalização , Hospitais Pediátricos , Malária/complicações , Malária/epidemiologia , Plasmodium falciparum/imunologia , Inconsciência/etiologia , Adolescente , Fatores Etários , Criança , Pré-Escolar , Feminino , Hospitais Públicos , Humanos , Incidência , Lactente , Malária/parasitologia , Malária/transmissão , Masculino , Morbidade , Plasmodium falciparum/isolamento & purificação , Prevalência , Estudos Retrospectivos , Uganda/epidemiologia
18.
BMC Infect Dis ; 20(1): 413, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32539801

RESUMO

BACKGROUND: Successful control programs have impeded local malaria transmission in almost all Gulf Cooperation Council (GCC) countries: Qatar, Bahrain, Kuwait, Oman, the United Arab Emirates (UAE) and Saudi Arabia. Nevertheless, a prodigious influx of imported malaria via migrant workers sustains the threat of local transmission. Here we examine the origin of imported malaria in Qatar, assess genetic diversity and the prevalence of drug resistance genes in imported Plasmodium falciparum, and finally, address the potential for the reintroduction of local transmission. METHODS: This study examined imported malaria cases reported in Qatar, between 2013 and 2016. We focused on P. falciparum infections and estimated both total parasite and gametocyte density, using qPCR and qRT-PCR, respectively. We also examined ten neutral microsatellites and four genes associated with drug resistance, Pfmrp1, Pfcrt, Pfmdr1, and Pfkelch13, to assess the genetic diversity of imported P. falciparum strains, and the potential for propagating drug resistance genotypes respectively. RESULTS: The majority of imported malaria cases were P. vivax, while P. falciparum and mixed species infections (P. falciparum / P. vivax) were less frequent. The primary origin of P. vivax infection was the Indian subcontinent, while P. falciparum was mostly presented by African expatriates. Imported P. falciparum strains were highly diverse, carrying multiple genotypes, and infections also presented with early- and late-stage gametocytes. We observed a high prevalence of mutations implicated in drug resistance among these strains, including novel SNPs in Pfkelch13. CONCLUSIONS: The influx of genetically diverse P. falciparum, with multiple drug resistance markers and a high capacity for gametocyte production, represents a threat for the reestablishment of drug-resistant malaria into GCC countries. This scenario highlights the impact of mass international migration on the reintroduction of malaria to areas with absent or limited local transmission.


Assuntos
Doenças Transmissíveis Importadas/transmissão , Resistência a Medicamentos/genética , Malária/transmissão , Plasmodium falciparum/genética , Doenças Transmissíveis Importadas/epidemiologia , Doenças Transmissíveis Importadas/parasitologia , Variação Genética , Genótipo , Humanos , Malária/epidemiologia , Malária/parasitologia , Carga Parasitária , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/genética , Plasmodium vivax/isolamento & purificação , Prevalência , Catar/epidemiologia
19.
Am J Trop Med Hyg ; 103(2): 558-560, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32553046

RESUMO

Rapid diagnostic tests (RDTs) play a critical role in malaria diagnosis and control. The emergence of Plasmodium falciparum parasites that can evade detection by RDTs threatens control and elimination efforts. These parasites lack or have altered genes encoding histidine-rich proteins (HRPs) 2 and 3, the antigens recognized by HRP2-based RDTs. Surveillance of such parasites is dependent on identifying false-negative RDT results among suspected malaria cases, a task made more challenging during the current pandemic because of the overlap of symptoms between malaria and COVID-19, particularly in areas of low malaria transmission. Here, we share our perspective on the emergence of P. falciparum parasites lacking HRP2 and HRP3, and the surveillance needed to identify them amid the COVID-19 pandemic.


Assuntos
Infecções por Coronavirus/epidemiologia , Testes Diagnósticos de Rotina/métodos , Malária Falciparum/diagnóstico , Plasmodium falciparum/isolamento & purificação , Pneumonia Viral/epidemiologia , África , Antígenos de Protozoários/análise , Betacoronavirus , Humanos , Malária Falciparum/epidemiologia , Pandemias , Proteínas de Protozoários/análise
20.
PLoS One ; 15(6): e0234263, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32525900

RESUMO

The current context of malaria elimination requires urgent development and implementation of highly sensitive and specific methods for prompt detection and treatment of malaria parasites. Such methods should overcome current delays in diagnosis, allow the detection of low-density infections and address the difficulties in accessing remote endemic communities. In this study, we assessed the performance of the RealAmp and malachite-green loop mediated isothermal amplification (MG-LAMP) methodologies, using microscopy and conventional nested-PCR as reference techniques. Both LAMP techniques were performed for Plasmodium genus, P. falciparum, and P. vivax identification using 136 whole blood samples collected from three communities located in the Peruvian Amazon basin. Turnaround time and costs of performing the LAMP assays were estimated and compared to that of microscopy and nested-PCR. Using nested-PCR as reference standard, we calculated the sensitivity, specificity and 95% confidence interval (CI) for all methods. RealAmp had a sensitivity of 92% (95% CI: 85-96.5%) and specificity of 100% (95% CI: 89.1-100%) for species detection; sensitivity and specificity of MG-LAMP were 94% (95% CI: 87.5-97.8%) and 100% (89.1-100%), respectively. Whereas microscopy showed 88.1% sensitivity (95% CI: 80.2-93.7%) and 100% specificity (95%: 89.1-100%). The turnaround time and costs of performing the LAMP assays were lower compared to those associated with nested-PCR but higher than those associated with microscopy. The two LAMP assays were shown to be more sensitive and simple to implement than microscopy. Both LAMP methodologies could be used as large-scale screening tests, but the MG-LAMP assay uses a simple, portable heat-block while the RealAmp requires a RealAmp machine or a real-time PCR machine. This makes the MG-LAMP an appropriate choice for malaria surveillance studies in endemic sites. Use of LAMP tests in active case detection of Plasmodium parasites could help to detect positive malaria cases early.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Compostos Organometálicos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/genética , Plasmodium vivax/isolamento & purificação , Temperatura , Adulto , Feminino , Humanos , Limite de Detecção , Malária Falciparum/diagnóstico , Malária Vivax/diagnóstico , Masculino , Plasmodium falciparum/fisiologia , Plasmodium vivax/fisiologia , Fatores de Tempo
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