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Background: Scientific information on the impact of malaria on the risk of developing type 2 diabetes mellitus (T2DM) after recovery from the coronavirus disease 2019 (COVID-19) is limited in the Ghanaian context. The purpose of this study was to examine the association between selected risk markers of T2DM in falciparum malaria patients post-COVID-19 or not at a tertiary hospital in Ghana. Methodology: This was a descriptive cross-sectional comparative study of 38-recovered COVID-19 adult participants with malaria and 40 unexposed COVID-19 adults with malaria at the Tamale Teaching Hospital, Ghana. Demographic, anthropometric and levels of glucose, insulin, C-reactive protein and lipid profiles were measured in the two groups of participants under fasting conditions. Parasitaemia was assessed microscopically but insulin resistance and beta-cell function were assessed by the homeostatic model. Results: The COVID-19 exposed participants were older (p=0.035) with lower parasitaemia (p=0.025) but higher mean levels of insulin, insulin resistance, and beta-cell function compared with their unexposed counterparts (p<0.05). Parasitaemia correlated positively with a number of the measured indices of diabetogenic risk markers in the COVID-19 exposed group only, and predicted (Adjusted R2=0.751; p=0.031) by beta-cell function, C-reactive protein and triglycerides with the model explaining about 75% of the observed variation. Parasitaemia could only be predicted (Adjusted R2=0.245; p=0.002) by C-reactive protein with the model explaining just about a quarter of the observed variation in the COVID-19 unexposed group. Insulin resistance and sub-optimal beta-cell function were detected in both groups of participants. Conclusion: Falciparum malaria is associated with risk markers for development of T2DM irrespective of COVID-19 exposure. Insulin resistance, inflammation and sub-optimal beta-cell secretory function may drive the risk. The observed diabetogenic risk is higher in the recovered COVID-19 participants.
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Humans , Male , Female , Malaria, Falciparum , Diabetes Mellitus, Type 2 , COVID-19 , Inflammation , Risk FactorsABSTRACT
Introducción. La resistencia de Plasmodium falciparum a diferentes fármacos antipalúdicos es un obstáculo para eliminar la enfermedad. El genotipo resistente de P. falciparum a la artemisinina puede evaluarse examinando los polimorfismos en el dominio de la hélice del gen Pfk13. La Organización Mundial de la Salud recomienda utilizar estas mutaciones como marcadores moleculares para detectar la resistencia a la artemisinina en países donde la malaria por P. falciparum es endémica. Objetivo. Identificar mutaciones relacionadas con la resistencia a artemisinina presentes en el dominio de la hélice del gen k13 de P. falciparum. Materiales y métodos. Mediante la detección pasiva de casos, se recolectaron 51 muestras positivas por microscopía para Plasmodium, provenientes de seis comunidades del distrito de Río Santiago en Condorcanqui, Amazonas. Se realizó la confirmación molecular de la especie mediante PCR en tiempo real y el dominio de la hélice del gen Pfk13 se amplificó y secuenció por electroforesis capilar. Las secuencias obtenidas se compararon con la cepa de referencia 3D7 de fenotipo silvestre. Resultados. Se confirmó un total de 51 muestras positivas para P. falciparum, provenientes de las comunidades de Ayambis, Chapiza, Palometa, Muchinguis, Alianza Progreso y Caterpiza. Después del alineamiento de las secuencias de ADN, se determinó que las muestras no presentaron mutaciones asociadas con resistencia en el gen K13. Discusión. Los resultados obtenidos son coherentes con estudios similares realizados en otros países de Sudamérica, incluyendo Perú. Estos datos proporcionan una línea base para la vigilancia molecular de resistencia a artemisinina en la región Amazonas y refuerzan la eficacia de la terapia combinada con artemisinina en esta área.
Introduction. Resistance of Plasmodium falciparum to different antimalarial drugs is an obstacle to disease elimination. The artemisinin-resistant genotype of P. falciparum can be assessed by examining polymorphisms in the helix domain of the Pfk13 gene. The World Health Organization recommends these mutations as molecular markers to detect artemisinin-resistant in countries where P. falciparum malaria is endemic. Objective. To identify artemisinin resistance-related mutations present in the helix domain of the P. falciparum k13 gene. Materials and methods. We collected a total of 51 samples through passive case detection, positive for Plasmodium by microscopy, from six communities in the district of Río Santiago in Condorcanqui, Amazonas. Molecular species confirmation was performed by real-time PCR and Pfk13 helix domain was amplified and sequenced by capillary electrophoresis. The obtained sequences were compared with the wild type 3D7 reference strain. Results. A total of 51 positive samples were confirmed for P. falciparum from the communities of Ayambis, Chapiza, Palometa, Muchinguis, Alianza Progreso and Caterpiza. DNA sequences alignment showed the absence of resistance-associated mutations in the k13 gene of the collected samples. Discussion. The obtained results are consistent with similar studies conducted in other South American countries, including Perú, so these data provide a baseline for artemisinin- resistance molecular surveillance in the Amazon region and reinforce the efficacy of artemisinin-based combination therapy in this area.
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Drug Resistance , Malaria , Peru , Plasmodium falciparum , Amazonian EcosystemABSTRACT
The aggregation of erythrocytes is an important mechanism for blood flow through the cardiovascular system. In malaria, this is complicated by infection caused by P. falciparum and is further complicated by the severity of parasitemia. Hence analysis of this micro-mechanism is essential to know the changes in blood not only in diseased conditions but also after artemisinin combination therapy (ASAQ) to alleviate suffering. For analysis purposes, aggregation of erythrocytes was determined by LED laser aggregometer, represented in terms of various parameters related to the changes in laser transmitted intensity. Formed aggregates are further analyzed by imaging and image-processing methods. For this study blood samples from young adults (18 – 40 years old) infected with P. falciparum (n= 80), without any other serious illness, were performed. These samples were selected based on the severity of parasitemia, and were divided into low (LP), medium1 (MP1), medium 2 (MP2), and high (HP) parasitemia. For three days, the selected individuals were treated with artemisinin-based combination therapy ASAQ (Artesunate 4 mg/kg and amodiaquine 10 mg base/ kg once a day). Healthy subjects (n=20) without any history of the disease were selected as a control group. The results, as obtained by various parameters, show a significant elevation of aggregation of erythrocytes (P< 0.05) in P. falciparum malaria with the increase of parasitemia level. There was a decrease in the aggregation after treatment on day four tending towards normal. Thus the current study shows the potential beneficial role of ASAQ on erythrocytes aggregation, which may contribute to reducing the harmful effects on various organs in P. falciparum-infected blood.
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Background: Malaria is a major health issue in tropical and subtropical areas. Out of all subtypes, Plasmodium falciparum (Pf) is the most dangerous form accounting for high mortality and morbidity. It is transmitted by infected female anopheles mosquitoes and infected blood transfusions. Aims and Objectives: The aim of the study is to establish correct diagnosis by direct microscopy, Immunochromatographic test (ICT), and molecular studies. Materials and Methods: This prospective study was conducted in the PG Department of Microbiology, SCB Medical College, Cuttack. Thick blood smears were drawn and then stained with Leishman’s stain to visualize falciparum rings. DNA was extracted from infected blood samples by phenol chloroform method with some modification as described by Sambrook and Russel for molecular analysis. Results: In the present study, 150 cases of malaria were analyzed. The male: female ratio was 1.7:1 and age ranged from 0 to 56 years. The Plasmodium vivax positivity was compared with thin smear to 21 (84%) in ICT, 100% both polymerase chain reaction (PCR) and loop mediated isothermal amplification assay (LAMP) assays followed by the Pf positivity as 76 (92.7%) in ICT, 82 (100%) both PCR and LAMP assays, respectively. The results obtained were statistically significant with P < 0.001. The PCR and LAMP showed 100% response to specificity and positive predictive value. Conclusion: The present study established the role of molecular tests such as PCR and LAMP are highly specific for diagnosis of Plasmodium species whereas they are more or less similar in sensitivity as compared to other diagnostic methods such as ICT and microscopy.
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Autoimmune hemolytic anemia (AIHA) has been rarely reported worldwide or from India as the underlying cause of anemia in malaria. We hereby present a case of complicated Plasmodium falciparum malaria with concomitant warm AIHA in a 31-year-old male. Direct Antiglobulin Test (DAT) was positive and elution studies showed pan-agglutination reaction. Clinico-hematological and serological follow-up of the patient was done post artesunate treatment until day 9. We suggest that it is important to establish the immune basis of anemia in malaria patients for guiding the treatment plan for the clinicians and providing packed red blood cell transfusion if required.
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Background & objectives: The spread of drug-resistant Plasmodium falciparum ( Pf) poses a serious threat to the control and elimination of malaria. The objective of this study was to detect the molecular biomarkers of antimalarial drug resistance in Pf in patients visiting a tertiary care hospital in Assam. Methods: Malaria was first detected in fever cases using microscopy and a rapid diagnostic test (RDT), and then confirmed using PCR. Pf chloroquine resistance transporter (Pfcrt), Pf multidrug resistance-1 (Pfmdr-1), and single-nucleotide polymorphisms linked to delayed parasite clearance after treatment with artemisinin MAL 10-688956 and MAL 13-1718319 and Kelch-13 propeller (PfK-13) genes were evaluated by PCR-restriction fragment length polymorphism (RFLP). Results: Sixty nine cases of malaria were found among 300 cases of fever. Of these, 54 were positive for Pf, 47 of which were confirmed by PCR. Pfcrt-K76T mutation was seen in 96.6 per cent and Pfmdr1-N86Y mutation in 84.2 per cent of cases. Mutation was not detected in MAL10 and MAL13 genes. Sequence analysis of Kelch-13 gene showed the presence of a novel mutation at amino acid position 675. Statistically, no significant association was found between the molecular biomarkers and demographic profile, clinical presentation and outcome of the cases. Interpretation & conclusions: Molecular surveillance is essential to assess the therapeutic efficacy of the drugs against circulating Pf isolates in Assam which are found to be highly resistant to CQ. The role of the new mutation found in the Kelch-13 gene in the development of artemisinin resistance in Assam needs to be thoroughly monitored in future research.
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Background & objectives: India targets malaria elimination by 2030 in a phased manner, so malaria’s assured diagnosis is crucial. Introduction of rapid diagnostic kits in India in 2010 has revolutionized malaria surveillance. The storage temperature of rapid diagnostic tests (RDTs), kit components and handling in transportations impact the results of RDTs. Therefore, quality assurance (QA) is required before it reaches end-users. The Indian Council of Medical Research-National Institute of Malaria Research (ICMR-NIMR) has a World Health Organization (WHO) recognized lot-testing laboratory facility to assure the quality of RDTs. Methods: The ICMR-NIMR receives RDTs from different manufacturing companies as well as various agencies such as National and State Programmes and Central Medical Services Society. The WHO standard protocol is followed to conduct all the tests, including long-term and post-dispatch testing. Results: A total of 323 lots tested during January 2014-March 2021 were received from different agencies. Amongst them, 299 lots passed the quality of test and 24 failed. In long-term testing, 179 lots were tested and only nine failed. A total of 7741 RDTs were received from end-users for post-dispatch testing of which 7540 qualified the QA test with a score of 97.4 per cent. Interpretation & conclusions: RDTs received for quality testing showed compliance with QA evaluation of malaria RDTs based on the protocol recommended by the WHO. However, continuous monitoring of the quality of RDTs is required under QA programme. Quality-assured RDTs have a major role, especially in areas where low parasitaemia of parasites persists.
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ABSTRACT The use of herbal tea with Artemisia annua by travelers and traditional communities in Africa has increased in recent years as a supposed form of malaria prophylaxis, although its use is not recommended due to lack of efficacy. The risk of severe malaria complications that can lead to death is real regarding said behavior, and awareness needs to be raised. We report a case of severe Plasmodium falciparum malaria imported in the Amazon rainforest by a traveler returning from Cameroon who treated himself with Artemisia annua herbal tea.
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BACKGROUND Cerebral malaria (CM) is a severe immunovasculopathy caused for Plasmodium falciparum infection, which is characterised by the sequestration of parasitised red blood cells (pRBCs) in brain microvessels. Previous studies have shown that some terpenes, such as perillyl alcohol (POH), exhibit a marked efficacy in preventing cerebrovascular inflammation, breakdown of the brain-blood barrier (BBB) and brain leucocyte accumulation in experimental CM models. OBJECTIVE To analyse the effects of POH on the endothelium using human brain endothelial cell (HBEC) monolayers co-cultured with pRBCs. METHODOLOGY The loss of tight junction proteins (TJPs) and features of endothelial activation, such as ICAM-1 and VCAM-1 expression were evaluated by quantitative immunofluorescence. Microvesicle (MV) release by HBEC upon stimulation by P. falciparum was evaluated by flow cytometry. Finally, the capacity of POH to revert P. falciparum-induced HBEC monolayer permeability was examined by monitoring trans-endothelial electrical resistance (TEER). FINDINGS POH significantly prevented pRBCs-induced endothelial adhesion molecule (ICAM-1, VCAM-1) upregulation and MV release by HBEC, improved their trans-endothelial resistance, and restored their distribution of TJPs such as VE-cadherin, Occludin, and JAM-A. CONCLUSIONS POH is a potent monoterpene that is efficient in preventing P. falciparum-pRBCs-induced changes in HBEC, namely their activation, increased permeability and alterations of integrity, all parameters of relevance to CM pathogenesis.
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@#Abstract: Objective To investigate the prevalence of C580Y mutation of kelch13 gene in the imported P. falciparum cases in Wuhan City, China, and to provide a reference basis for the prevention and treatment of imported falciparum malaria. Methods From 2009 to 2015, blood samples were collected from returnees who infected with P. falciparum in endemic areas of Africa and Southeast Asia in Wuhan City. The P. falciparum DNA was extracted from the blood samples, and kelch13 gene was amplified by loop-mediated isothermal amplification (LAMP), and the distribution of C580Y mutation was analyzed. Results C580Y mutation was detected in 16 of the 208 cases tested by LAMP. No mutations were detected in 69 cases of imported falciparum malaria from Africa during 2009-2012, while 13 cases of the C580Y mutation were detected in 114 cases from 2013 to 2015, with a mutation rate of 11.4%. The mutation rate in South Africa, West Africa, and Central Africa was 12.5%, 9.6%, and 19.0%, respectively, with no mutations detected in cases from North Africa and East Africa. Among the 25 cases of falciparum malaria from Southeast Asia between 2009 and 2013, three cases were positive for the C580Y mutation, all from Myanmar, with a mutation rate of 14.3% (3/21) in Myanmar and 12.0% (3/25) in Southeast Asia. There was no significant difference in the mutation rate between Africa and Southeast Asia after 2013 (P>0.05). Conclusions Our findings highlight the varying degrees of C580Y mutations of kelch13 gene in imported P. falciparum cases in Wuhan and suggest the need for enhanced monitoring and evaluation of related resistance genes.
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Objective To investigate the prevalence of single nucleotide polymorphisms (SNPs) of artemisinin resistance-related Pfubp1 and Pfap2mu genes in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea, so as to to provide baseline data for the formulation of malaria control strategies in Bioko Island. Methods A total of 184 clinical blood samples were collected from patients with P. falciparum malaria in Bioko Island, Equatorial Guinea from 2018 to 2020, and genomic DNA was extracted. The Pfubp1 and Pfap2mu gene SNPs of P. falciparum were determined using a nested PCR assay and Sanger sequencing, and the gene sequences were aligned. Results There were 159 wild-type P. falciparum isolates (88.83%) from Bioko Island, Equatorial Guinea, and 6 SNPs were identified in 20 Pfubp1-mutant P. falciparum isolates (11.17%), in which 4 non-synonymous mutations were detected, including E1516G, K1520E, D1525E, E1528D. There was only one Pfubp1gene mutation site in 19 Pfubp1-mutant P. falciparum isolates (95.00%), in which non-synonymous mutations accounted for 68.42% (13/19). D1525E and E1528D were identified as major known epidemic mutation sites in the Pfubp1 gene associated with resistance to artemisinin-based combination therapies (ACTs). At amino acid position 1525, there were 178 wild-type P. falciparum isolates (99.44%) and 1 mutant isolate (0.56%), with such a mutation site identified in blood samples in 2018, and at amino acid position 1528, there were 167 wild-type P. falciparum isolates (93.30%) and 12 mutant isolates (6.70%). The proportions of wild-type P. falciparum isolates were 95.72% (134/140), 79.25% (126/159) and 95.83% (161/168) in the target amplification fragments of the three regions in the Pfap2mu gene (Pfap2mu-inner1, Pfap2mu-inner2, Pfap2mu-inner3), respectively. There were 16 different SNPs identified in all successfully sequenced P. falciparum isolates, in which 7 non-synonymous mutations were detected, including S160N, K199T, A475V, S508G, I511M, L595F, and Y603H. There were 7 out of 43 Pfap2mu-mutant P. falciparum isolates (16.28%) that harbored only one gene mutation site, in which non-synonymous mutations accounted for 28.57% (2/7). For the known delayed clearance locus S160N associated with ACTs, there were 143 wild-type (89.94%) and 16 Pfap2mu-mutant P. falciparum isolates (10.06%). Conclusions Both Pfubp1 and Pfap2mu gene mutations were detected in P. falciparum isolates from Bioko Island, Equatorial Guinea from 2018 to 2020, with a low prevalence rate of Pfubp1 gene mutation and a high prevalence rate of Pfap2mu gene mutation. In addition, new mutation sites were identified in the Pfubp1 (E1504E and K1520E) and Pfap2mu genes (A475V and S508G).
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Objective To establish a fluorescent assay for rapid detection of Plasmodium falciparum based on recombinaseaided amplification (RAA) and CRISPR-Cas12a system,and to preliminarily evaluate the diagnostic efficiency of this system.. Methods The 18S ribosomal RNA (rRNA) gene of P. falciparum was selected as the target sequence, and three pairs of RAA primers and CRISPR-derived RNA (crRNA) were designed and synthesized. The optimal combination of RAA primers and crRNA was screened and the reaction conditions of the system were optimized to create a fluorescent RAA/CRISPR-Cas12a system. The plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 was generated, and diluted into concentrations of 1 000, 100, 10, 1 copy/μL for the fluorescent RAA/CRISPR-Cas12a assay, and its sensitivity was evaluated. The genomic DNA from P. vivax, P. malariae, P. ovum, hepatitis B virus, human immunodeficiency virus and Treponema pallidum was employed as templates for the fluorescent RAA/CRISPR-Cas12a assay, and its specificity was evaluated. Fifty malaria clinical samples were subjected to the fluorescent RAA/CRISPR-Cas12a assay and nested PCR assay, and the consistency between two assays was compared. In addition, P. falciparum strain 3D7 was cultured in vitro. Then, the culture was diluted into blood samples with parasite densities of 1 000, 500, 200, 50, 10 parasites/μL with healthy volunteers’ O-positive red blood cells for the RAA/CRISPR-Cas12a assay, and the detection efficiency was tested. Results The Pf-F3/Pf-R3/crRNA2 combination, 2.5 μL as the addition amount of B buffer, 40 min as the RAA reaction time, 37 °C as the reaction temperature of the CRISPR-Cas12a system were employed to establish the fluorescent RAA/CRISPR-Cas12a system. Such a system was effective to detect the plasmid containing 18S rRNA gene of the P. falciparum strain 3D7 at a concentration of 1 copy/μL, and presented fluorescent signals for detection of P. falciparum, but failed to detect P. ovum, P. malariae, P. vivax, T. pallidum, hepatitis B virus or human immunodeficiency virus. The fluorescent RAA/CRISPR-Cas12a system and nested PCR assay showed completely consistent results for detection of 50 malaria clinical samples (kappa = 1.0, P < 0.001). Following 6-day in vitro culture of the P. falciparum strain 3D7, 10 mL cultures were generated and the fluorescent RAA/CRISPR-Cas12a system showed the minimal detection limit of 50 parasites/μL. Conclusion The fluorescent RAA/CRISPR-Cas12a system is rapid, sensitive and specific for detection of P. falciparum, which shows promising value for rapid detection and risk monitoring of P. falciparum.
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@#Malaria, caused by the unicellular Apicomplexan protozoa of the genus Plasmodium, is an infectious disease transmitted via female Anopheles mosquitoes. The sexual stage (gametocytes) of malaria parasites is the key to the transmission of parasites from vertebrate hosts to mosquitoes, representing critical bottleneck of the parasite life cycle. This study has established a systematic computational pipeline to achieve the genome-wide in silico analysis and find 708 novels potentially indispensable genes for gametocyte development, consisting of 644 protein coding genes, 56 ncRNA genes and 8 pseudogenes, with a total of 191 genes in the transmembrane, 29 protein coding genes to be exported proteins, and 58 genes in apicoplast regions. Furthermore, Gene Ontology analysis showed that the largest cluster was cellular processes with nucleus and cytosol highest, followed by molecular function with binding and oxidoreductase activities abundant. Meanwhile, when a text searched, using PlasmoDB, there were 300 genes with annotations of “putative”, and 196 genes with annotations of “unknown function”. These data would be helpful to provide potential targets for effective malaria transmission-blocking strategies.
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@#Malaria is a life-threatening disease caused by protozoan Plasmodium species. Plasmodium falciparum is the deadliest species. Reducing and eliminating malaria burden are linked to most of the Sustainable Development Goals (SDG), central to SDG3 targeting the end of malaria by 2030. This study was aimed at assessing the Management of malaria and prevalence of P. falciparum kelch-13 among febrile patients in selected Government Hospitals in Nigeria. Malaria patients (399) attending outpatient clinics of the Hospitals between August, 2019 and January, 2021, were enlisted in the study, following ethical approval and informed consents. Blood (5mL) was collected from patients for microscopic and molecular investigation of malaria parasite. DNA extraction, PCR amplification, BLAST, and alignment were performed. Plasmodium resistance to Artemether/lumefantrine was determined by PCR amplification of extracted DNA using Kelch-13 gene primer. Data obtained were subjected to One-way Analysis of Variance and Linear Regression. The VapA gene primer amplified 55 (68.75%) out of the 80 DNA extracts tested. Twenty-five strains of P. falciparum belonging to 3 clades phylogenetically were identified and they showed evolutionary relationships with others. Plasmodium falciparum resistant Kelch-13 gene was detected in 70% of the isolates. This study observed a high prevalence of resistant gene to ACT drugs in the study area. Monitoring the effectiveness of ACTs must be done routinely to ensure timely changes in National treatment policies.
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@#Abstract: Transfection of Plasmodium falciparum is helpful to study the function of its genes, such as drug resistance. However, transgenic manipulation has been very challenging, mainly due to the high A/T base sequence structure (A+T content of about 82%) and low transfection efficiency of the Plasmodium genome. Electroporation-based transfection of Plasmodium falciparum has been successfully applied in the study of certain genes, and electroporation by preloading is currently the preferred method for introducing foreign DNA into Plasmodium falciparum. The site-directed editing of Plasmodium genes mostly adopts the method of two-plasmid transfection. It is generally believed that successful transfection of Plasmodium requires a large amount of high-purity plasmid DNA and an accurate transfection system. In addition to the evaluation of the current commonly used electrotransfection methods, this paper also introduces a new transfection method, namely lyse-reseal erythrocytes for transfection (LyRET). This paper also review the role of factors such as plasmid DNA concentration, the use of transfection reagents, the setting of transfection parameters, the addition of fresh red blood cells, and the markers of successful transfection in improving the success rate and efficiency of Plasmodium transfection, in the hope of providing a reference for study in this field.
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@#Abstract: Objective To analyze the laboratory indexes of patients infected with malaria patients and COVID-19, so as to provide reliable evidence for the diagnosis of mixed infection of both. Methods The routine clinical laboratory items such as routine blood, biochemistry and lymphocyte subsets were tested in three cases of COVID-19 complicated with falciparum malaria who admitted to Guangzhou Eighth People's Hospital Affiliated to Guangzhou Medical University from July to December 2020 were tested. Laboratory data were stage-wise analyzed in conjunction with changes in the course of disease. Results Three patients confirmed COVID-19 infection recruited all had malaria infection history. Fever, headache, and other symptoms emerged on the 4rd to 11th day after admission. Malaria parasite was detected by malaria parasite antigen testing and blood smear testing, and all three patients had re-ignition of malaria after being confirmed COVID-19 infection. In the early stage of malaria relapse, lymphocytes decreased, CRP and SAA increased, and gradually returned to normal level after antimalarial treatment. Interestingly, we only found one patient at the initial stage of malaria detection showed PLT decreased, no other unnormal changes in other routine blood results (WBC, ESO) and liver function results (ALT, AST, GGT, TBIL, DBIL, CG) were found from the beginning to end course of the disease. Conclusion COVID-19 infection may promote the resurgence of malaria, so the relapse of malaria should be monitored especially for the patient with malaria infection history who begin to develop fever and other symptoms a few days after the diagnosis of COVID-19. The inflammatory indicators would be worth able as an auxiliary judgment basis for the effective treatment of the two combined infection.
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Abstract Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.
Resumo A resistência do Plasmodium falciparum à cloroquina (CQ) é uma causa significativa de mortalidade e morbidade em todo o mundo. Há uma escassez de dados documentados sobre a prevalência de haplótipos mutantes CQ-resistentes dos genes Pfcrt e Pfmdr1 da guerra endêmica da malária em áreas tribais administradas pelo governo federal do Paquistão. O objetivo deste estudo foi investigar a prevalência de resistência a CQ de P. falciparum nesta área. Isolados clínicos foram coletados entre maio de 2017 e maio de 2018 nas agências do Waziristão do Norte e do Waziristão do Sul da Área de Ensaio Administrada Federalmente. Posteriormente, esfregaços de sangue corados com Giemsa foram examinados para detectar Plasmodium falciparum. A extração do DNA da malária foi feita a partir de amostras de P. falciparum positivas para microscopia, e as infecções por P. falciparum foram confirmadas por nested PCR (visando genes de ácido ribonucleico ribossômico de subunidade pequena de Plasmodium (ssrRNA)). Todas as amostras de P. falciparum confirmadas por PCR foram sequenciadas por pirosequenciamento para descobrir a mutação no gene Pfcrt no códon K76T e em pfmdr1 nos códons N86Y, Y184F, N1042D e D1246Y. De 121 microscopias de casos positivos de P. falciparum, 109 amostras foram positivas para P. falciparum por nested PCR. A mutação Pfcrt K76T foi encontrada em 96% dos isolados, a mutação Pfmdr1 N86Y foi observada em 20% e 11% abrigou a mutação Y184F. Todas as amostras eram do tipo selvagem para o códon N1042D e D1246Y de Pfmdr1. No FATA, Paquistão, a frequência do alelo resistente 76T permaneceu alta apesar da remoção de CQ. No entanto, as descobertas atuais do estudo sugerem a fixação completa do genótipo resistente a CQ de P. falciparum na área de estudo.
Subject(s)
Plasmodium falciparum/genetics , Antimalarials/pharmacology , Pakistan , Membrane Transport Proteins/genetics , Drug Resistance/genetics , Protozoan Proteins/genetics , Chloroquine/pharmacology , Multidrug Resistance-Associated Proteins/genetics , AllelesABSTRACT
Abstract Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.
Resumo A resistência do Plasmodium falciparum à cloroquina (CQ) é uma causa significativa de mortalidade e morbidade em todo o mundo. Há uma escassez de dados documentados sobre a prevalência de haplótipos mutantes CQ-resistentes dos genes Pfcrt e Pfmdr1 da guerra endêmica da malária em áreas tribais administradas pelo governo federal do Paquistão. O objetivo deste estudo foi investigar a prevalência de resistência a CQ de P. falciparum nesta área. Isolados clínicos foram coletados entre maio de 2017 e maio de 2018 nas agências do Waziristão do Norte e do Waziristão do Sul da Área de Ensaio Administrada Federalmente. Posteriormente, esfregaços de sangue corados com Giemsa foram examinados para detectar Plasmodium falciparum. A extração do DNA da malária foi feita a partir de amostras de P. falciparum positivas para microscopia, e as infecções por P. falciparum foram confirmadas por nested PCR (visando genes de ácido ribonucleico ribossômico de subunidade pequena de Plasmodium (ssrRNA)). Todas as amostras de P. falciparum confirmadas por PCR foram sequenciadas por pirosequenciamento para descobrir a mutação no gene Pfcrt no códon K76T e em pfmdr1 nos códons N86Y, Y184F, N1042D e D1246Y. De 121 microscopias de casos positivos de P. falciparum, 109 amostras foram positivas para P. falciparum por nested PCR. A mutação Pfcrt K76T foi encontrada em 96% dos isolados, a mutação Pfmdr1 N86Y foi observada em 20% e 11% abrigou a mutação Y184F. Todas as amostras eram do tipo selvagem para o códon N1042D e D1246Y de Pfmdr1. No FATA, Paquistão, a frequência do alelo resistente 76T permaneceu alta apesar da remoção de CQ. No entanto, as descobertas atuais do estudo sugerem a fixação completa do genótipo resistente a CQ de P. falciparum na área de estudo.
ABSTRACT
Abstract Military conflicts have been significant obstacles in detecting and treating infectious disease diseases due to the diminished public health infrastructure, resulting in malaria endemicity. A variety of violent and destructive incidents were experienced by FATA (Federally Administered Tribal Areas). It was a struggle to pursue an epidemiological analysis due to continuing conflict and Talibanization. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai agencies from May 2017 to May 2018. For Giemsa staining, full blood EDTA blood samples have been collected from symptomatic participants. Malaria-positive microscopy isolates were spotted on filter papers for future Plasmodial molecular detection by nested polymerase chain reaction (nPCR) of small subunit ribosomal ribonucleic acid (ssrRNA) genes specific primers. Since reconfirming the nPCR, a malariometric study of 762 patients found 679 positive malaria cases. Plasmodium vivax was 523 (77%), Plasmodium falciparum 121 (18%), 35 (5%) were with mixed-species infection (P. vivax plus P. falciparum), and 83 were declared negative by PCR. Among the five agencies of FATA, Khyber agency has the highest malaria incidence (19%) with followed by P. vivax (19%) and P. falciparum (4.1%). In contrast, Kurram has about (14%), including (10.8%) P. vivax and (2.7%) P. falciparum cases, the lowest malaria epidemiology. Surprisingly, no significant differences in the distribution of mixed-species infection among all five agencies. P. falciparum and P. vivax were two prevalent FATA malaria species in Pakistans war-torn area. To overcome this rising incidence of malaria, this study recommends that initiating malaria awareness campaigns in school should be supported by public health agencies and malaria-related education locally, targeting children and parents alike.
Resumo Os conflitos militares têm sido obstáculos significativos na detecção e tratamento de doenças infecciosas devido à diminuição da infraestrutura de saúde pública, resultando na endemicidade da malária. Uma variedade de incidentes violentos e destrutivos foi vivida pelas FATA (áreas tribais administradas pelo governo federal). Foi uma luta buscar uma análise epidemiológica devido ao conflito contínuo e à talibanização. Isolados clínicos foram coletados de agências Bajaur, Mohmand, Khyber e Orakzai, de maio de 2017 a maio de 2018. Para a coloração de Giemsa, amostras de sangue completo com EDTA foram coletadas de participantes sintomáticos. Isolados de microscopia positivos para malária foram colocados em papéis de filtro para futura detecção molecular plasmódica por reação em cadeia da polimerase aninhada (nPCR) de primers específicos de genes de subunidade ribossômica de ácido ribonucleico (ssrRNA). Desde a reconfirmação do nPCR, um estudo malariométrico de 762 pacientes encontrou 679 casos positivos de malária. Plasmodium vivax foi 523 (77%), Plasmodium falciparum 121 (18%), 35 (5%) eram com infecção de espécies mistas (P. vivax mais P. falciparum) e 83 foram declarados negativos por PCR. Entre as cinco agências da FATA, a agência Khyber tem a maior incidência de malária (19%), seguida por P. vivax (19%) e P. falciparum (4,1%). Em contraste, Kurram tem cerca de 14%, incluindo 10,8% casos de P. vivax e 2,7% P. falciparum, a epidemiologia de malária mais baixa. Surpreendentemente, não há diferenças significativas na distribuição da infecção de espécies mistas entre todas as cinco agências. P. falciparum e P. vivax foram duas espécies prevalentes de malária FATA na área devastada pela guerra no Paquistão. Para superar essa incidência crescente de malária, este estudo recomenda que o início de campanhas de conscientização sobre a malária na escola deve ser apoiado por agências de saúde pública e educação relacionada com a malária localmente, visando crianças e pais.
ABSTRACT
Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ‑resistant genotype in the study area.
A resistência do Plasmodium falciparum à cloroquina (CQ) é uma causa significativa de mortalidade e morbidade em todo o mundo. Há uma escassez de dados documentados sobre a prevalência de haplótipos mutantes CQ-resistentes dos genes Pfcrt e Pfmdr1 da guerra endêmica da malária em áreas tribais administradas pelo governo federal do Paquistão. O objetivo deste estudo foi investigar a prevalência de resistência a CQ de P. falciparum nesta área. Isolados clínicos foram coletados entre maio de 2017 e maio de 2018 nas agências do Waziristão do Norte e do Waziristão do Sul da Área de Ensaio Administrada Federalmente. Posteriormente, esfregaços de sangue corados com Giemsa foram examinados para detectar Plasmodium falciparum. A extração do DNA da malária foi feita a partir de amostras de P. falciparum positivas para microscopia, e as infecções por P. falciparum foram confirmadas por nested PCR (visando genes de ácido ribonucleico ribossômico de subunidade pequena de Plasmodium (ssrRNA)). Todas as amostras de P. falciparum confirmadas por PCR foram sequenciadas por pirosequenciamento para descobrir a mutação no gene Pfcrt no códon K76T e em pfmdr1 nos códons N86Y, Y184F, N1042D e D1246Y. De 121 microscopias de casos positivos de P. falciparum, 109 amostras foram positivas para P. falciparum por nested PCR. A mutação Pfcrt K76T foi encontrada em 96% dos isolados, a mutação Pfmdr1 N86Y foi observada em 20% e 11% abrigou a mutação Y184F. Todas as amostras eram do tipo selvagem para o códon N1042D e D1246Y de Pfmdr1. No FATA, Paquistão, a frequência do alelo resistente 76T permaneceu alta apesar da remoção de CQ. No entanto, as descobertas atuais do estudo sugerem a fixação completa do genótipo resistente a CQ de P. falciparum na área de estudo.