Detection Of Plasmodium Parasites Research Articles

Comparison of Warmstart colorimetric LAMP using heat-treated samples with two Plasmodium-detection methods in Ekiti state, southwestern Nigeria

Background: Malaria remains a deadly disease responsible for numerous deaths worldwide, especially in tropi-cal developing countries. Widely used methods for diagnosis include microscopy, which is laborious, lengthy, and error-prone, and rapid diagnostic tests (RDT), which have recently faced limitations on accuracy. Molecular methods like the polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) have emerged as accurate, sensitive, and rapid ways of detecting the disease. While PCR requires expensive equip-ment, LAMP is less expensive to perform. However, contemporary malaria LAMP still requires DNA extraction kits from patient samples. DNA extraction by heating can be used to lower the cost, time, and level of training required from end-users to perform LAMP in low-resource settings. Methods: Blood samples were screened for Plasmodium parasites using microscopy, followed by a commonly used RDT kit. Heat treatment was used for quick DNA extraction from the blood samples, and LAMP primer sets were then used together with Warmstart colorimetric LAMP reagents for isothermal DNA amplification. Agarose gel electrophoresis was used to confirm that the colorimetric observations in LAMP reactions were due to successful DNA amplification. The data obtained were analysed in GraphPad Prism and subjected to chi-square tests with 95% confidence level. Results: Warmstart colorimetric LAMP using heat-treated blood samples achieved successful DNA amplifica-tion and detected Plasmodium parasites in 43.3% of samples, compared to 40% and 30% Plasmodium parasite detection yielded by antigen RDT kit and microscopy respectively. However, the difference between the percent-ages obtained using the different methods was not statistically significant. LAMP approach further enabled the identification/discrimination of different Plasmodium species. Conclusion: The Warmstart LAMP using heat-treated samples can be used as a rapid, cheaper, and easier-to-perform molecular method for the detection and species-level differentiation of malaria parasites.

The millennial dynamics of malaria in the mediterranean basin: documenting Plasmodium spp. on the medieval island of Corsica.

The lack of well-preserved material upon which to base the paleo-microbiological detection of Plasmodium parasites has prevented extensive documentation of past outbreaks of malaria in Europe. By trapping intact erythrocytes at the time of death, dental pulp has been shown to be a suitable tissue for documenting ancient intraerythrocytic pathogens such as Plasmodium parasites. Total DNA and proteins extracted from 23 dental pulp specimens collected from individuals exhumed from the 9th to 13th century archaeological site in Mariana, Corsica, were analyzed using open-mind paleo-auto-immunohistochemistry and direct metagenomics, Plasmodium-targeting immunochromatography assays. All experiments incorporated appropriate negative controls. Paleo-auto-immunohistochemistry revealed the presence of parasites Plasmodium spp. in the dental pulp of nine teeth. A further immunochromatography assay identified the presence of at least one Plasmodium antigen in nine individuals. The nine teeth, for which the PfHRP-2 antigen specific of P. falciparum was detected, were also positive using paleo-autoimmunohistochemistry and metagenomics. Dental pulp erythrocytes proved to be suitable for the direct paleomicrobiology documentation of malaria in nine individuals buried in medieval Corsica, in agreement with historical data. This provides additional information on the millennial dynamics of Plasmodium spp. in the Mediterranean basin.

IMAGING: a novel automated system for malaria diagnosis by using artificial intelligence tools and a universal low-cost robotized microscope

IntroductionMalaria is one of the most prevalent infectious diseases in sub-Saharan Africa, with 247 million cases reported worldwide in 2021 according to the World Health Organization. Optical microscopy remains the gold standard technique for malaria diagnosis, however, it requires expertise, is time-consuming and difficult to reproduce. Therefore, new diagnostic techniques based on digital image analysis using artificial intelligence tools can improve diagnosis and help automate it.MethodsIn this study, a dataset of 2571 labeled thick blood smear images were created. YOLOv5x, Faster R-CNN, SSD, and RetinaNet object detection neural networks were trained on the same dataset to evaluate their performance in Plasmodium parasite detection. Attention modules were applied and compared with YOLOv5x results. To automate the entire diagnostic process, a prototype of 3D-printed pieces was designed for the robotization of conventional optical microscopy, capable of auto-focusing the sample and tracking the entire slide.ResultsComparative analysis yielded a performance for YOLOv5x on a test set of 92.10% precision, 93.50% recall, 92.79% F-score, and 94.40% mAP0.5 for leukocyte, early and mature Plasmodium trophozoites overall detection. F-score values of each category were 99.0% for leukocytes, 88.6% for early trophozoites and 87.3% for mature trophozoites detection. Attention modules performance show non-significant statistical differences when compared to YOLOv5x original trained model. The predictive models were integrated into a smartphone-computer application for the purpose of image-based diagnostics in the laboratory. The system can perform a fully automated diagnosis by the auto-focus and X-Y movements of the robotized microscope, the CNN models trained for digital image analysis, and the smartphone device. The new prototype would determine whether a Giemsa-stained thick blood smear sample is positive/negative for Plasmodium infection and its parasite levels. The whole system was integrated into the iMAGING smartphone application.ConclusionThe coalescence of the fully-automated system via auto-focus and slide movements and the autonomous detection of Plasmodium parasites in digital images with a smartphone software and AI algorithms confers the prototype the optimal features to join the global effort against malaria, neglected tropical diseases and other infectious diseases.

Natural Plasmodium falciparum Infection Stimulates Human Antibodies to MSP1 Epitopes Identified in Mice Infection Models upon Non-Natural Modified Peptidomimetic Vaccination.

(1) Background: Malaria, a vector-borne infectious disease, is caused by parasites of the Plasmodium genus, responsible for increased extreme morbidity and mortality rates. Despite advances in approved vaccines, full protection has not yet been achieved upon vaccination, thus the development of more potent and safe immuno-stimulating agents for malaria prevention is a goal to be urgently accomplished. We have focused our research on a strategy to identify Plasmodium spp. epitopes by naturally acquired human antibodies and rodent malaria infection models immunized with site-directed non-natural antigens. (2) Methods: Some predictive algorithms and bioinformatics tools resembling different biological environments, such as phagosome-lysosome proteolytic degradation, affinity, and the high frequency of malaria-resistant and -sensitive HLA-II alleles were regarded for the proper selection of epitopes and potential testing. Each epitope's binding profile to both host cells and HLA-II molecules was considered for such initial screening. (3) Results: Once selected, we define each epitope-peptide to be synthesized in terms of size and hydrophobicity, and introduced peptide-bond surrogates and non-natural amino acids in a site-directed fashion, and then they were produced by solid-phase peptide synthesis. Molecules were then tested by their antigenic and immunogenic properties compared to human sera from Colombian malaria-endemic areas. The antigenicity and protective capacity of each epitope-peptide in a rodent infection model were examined. The ability of vaccinated mice after being challenged with P. berghei ANKA and P. yoelii 17XL to control malaria led to the determination of an immune stimulation involving Th1 and Th1/Th2 mechanisms. In silico molecular dynamics and modeling provided some interactions insights, leading to possible explanations for protection due to immunization. (4) Conclusions: We have found evidence for proposing MSP1-modified epitopes to be considered as neutralizing antibody stimulators that are useful as probes for the detection of Plasmodium parasites, as well as for sub-unit components of a site-directed designed malaria vaccine candidate.

Prevalence of simian malaria among macaques in Malaysia (2000-2021): A systematic review.

BackgroundThe aim of Malaysia to eliminate malaria nationwide by 2020 seems need to be prolonged. Whilst Malaysia has successfully eliminated human malaria transmission, simian malaria parasites such as Plasmodium knowlesi, P. cynomolgi, P. inui and P. cynomolgi are the emerging cause of malaria in humans. The epidemiological study of simian malaria in primates provides useful information in identifying the risk of human-macaques Plasmodium infection.Methodology/Principal findingsThis study was performed to gather all available data in terms of simian malaria epidemiology study among macaques in Malaysia over the last two decades. This systematic review was conducted according to the PRISMA guidelines to select appropriate articles as references. Data searches were performed through international databases such as Google Scholar, PubMed, CrossRef, Scopus, Web of Science and Science Direct for original articles published from 2000 until 2021. The review identified seven simian malaria epidemiology studies in Malaysia over the 20-year study period. Most studies were conducted in Peninsular Malaysia (5/7; 71%) followed by East Malaysia (2/7; 29%). All studies showed positive detection of Plasmodium parasites in macaques. The most prevalent Plasmodium species in macaques was P. inui (49.27%) and the least prevalent was P. fieldi (4.76%). The prevalence of simian malaria was higher in East Malaysia compared to Peninsular Malaysia. The mono, dual and triple infection types were the most common among macaques.Conclusion/SignificanceThe non-human primates like macaques are the reservoir of simian plasmodium in Malaysia. Hence, the study of host epidemiology is an important insight to public health management as there is a high occurrence of simian malaria in Malaysia. The right measurement can be taken as well to prevent the transmission of simian malaria from macaques to humans.

Blood meal profile and positivity rate with malaria parasites among different malaria vectors in Sudan

BackgroundMalaria is a life-threatening public health problem globally with particularly heavy burden in the sub-Saharan Africa including Sudan. The understanding of feeding preference of malaria vectors on different hosts is a major challenge for hindering the transmission cycle of malaria. In this study, blood meals taken by blood-fed Anopheles mosquitoes collected from the field in malaria endemic areas of Sudan were analysed for source of blood meal and malaria parasite presence.MethodsAnopheles mosquitoes were collected from different regions in Sudan: Khartoum state, Sennar state, Northern state, and El Gedarif state between September 2020 and February 2021. Anopheles mosquitoes were collected using the standard pyrethrum spray catch and back-pack aspirator. Mosquito samples were sorted and morphologically identified to species level using international identification keys. Morphologically identified mosquito species were also confirmed using PCR. Genomic DNA was extracted from mosquitoes for molecular identification of blood meal source and parasite detection. The presence of Plasmodium species DNA in each mosquito sample was investigated using semi-nested PCR. Frequency of each blood meal source, Anopheles mosquito vector, and malaria parasite detected was calculated. Positivity rate of each fed female Anopheles mosquito was calculated for each species.ResultsA total of 2132 Anopheles mosquitoes were collected. 571 (26.8%) were males and 1561 (73.2%) were females classified based on their abdominal status into 1048 (67.1%) gravid, 274 (17.6%) fed, and 239 (15.3%) unfed females. Among the blood fed Anopheles mosquitoes, 263 (96.0%) were morphologically identified and confirmed using PCR to Anopheles arabiensis, 9 (3.3%) to Anopheles stephensi, and 2 (0.7%) to Anopheles rufipes. Of 274 blood-fed An. arabiensis, 68 (25.9%) fed on mixed blood meals from human and cattle, 8 (3.0%) fed on cattle and goat, and 13 (4.8%) fed on human, cattle and goat. For single blood meal sources, 70 (26.6%) fed on human, 95 (36.1%) fed on cattle, 8 (3.0%) fed on goat, and 1 (0.4%) fed on dog. While An. rufipes and An. stephensi fed on dog (2; 0.75%) and cattle (9; 3.3%), respectively. Plasmodium parasite detection in the blood meals showed that 25/274 (9.1%) An. arabiensis meals were positive for Plasmodium vivax and 19/274 (6.9%) An. arabiensis meals were positive for Plasmodium falciparum. The rate of positivity of An. arabiensis with any Plasmodium species was 16.7%. However, the positivity rate with P. falciparum only was 7.2%, while P. vivax was 9.5%. Both An. rufipes and An. stephensi were having positivity rates of 0.0% each.ConclusionsThis study which was mainly on blood-fed Anopheles mosquitoes showed a diversity in the type of diet from human, cattle, and goat. Anopheles mosquitoes especially An. arabiensis in Sudan, are opportunistic blood feeders and can feed broadly on both human and cattle. The application of blood meal identification is not only important in malaria vector epidemiological surveillance but also is very useful in areas where arthropods exhibit zoophilic feeding behaviour for mammals.

Assessing Anopheles vector species diversity and transmission of malaria in four health districts along the borders of C\xf4te d\u2019Ivoire

BackgroundAlthough malaria and Anopheles mosquito vectors are highly prevalent in Côte d’Ivoire, limited data are available to help understand the malaria vector density and transmission dynamics in areas bordering the country. To address this gap, the Anopheles mosquito species diversity, the members of the Anopheles gambiae complex and the transmission of malaria were assessed in four health districts along the borders of Côte d’Ivoire.MethodsFrom July 2016 through December 2016 and July 2017 through December 2017, adult Anopheles mosquitoes were collected in four health districts of Côte d’Ivoire (Aboisso, Bloléquin, Odienné and Ouangolodougou) using standardized window exit trap (WET) and pyrethrum knockdown spray collection (PSC) methods. The collected mosquitoes were identified morphologically at species level and the members of the An. gambiae complex were separated using short interspersed nuclear element-based polymerase chain reaction (SINE-PCR). Anopheles gambiae sensu lato (s.l.), Anopheles funestus s.l. and Anopheles nili specimens were analysed for malaria Plasmodium parasite detection using the cytochrome oxidase I gene (COX-I), and malaria prevalence among human population through local Ministry of Health (MoH) statistical yearbooks.ResultsA total of 281 female Anopheles were collected in Aboisso, 754 in Bloléquin, 1319 in Odienné and 2443 in Ouangolodougou. Seven Anopheles species were recorded including An. gambiae s.l. (94.8–99.1%) as the main vector, followed by An. funestus s.l. (0.4–4.3%) and An. nili (0–0.7%). Among An. gambiae s.l., Anopheles coluzzii represented the predominant species in Aboisso (89.2%) and Bloléquin (92.2%), while An. gambiae sensu stricto (s.s.) was the major species in Odienné (96.0%) and Ouangolodougou (94.2%). The Plasmodium sporozoite infection rate in An. gambiae s.l. was highest in Odienné (11.0%; n = 100) followed by Bloléquin (7.8%, n = 115), Aboisso (3.1%; n = 65) and Ouangologoudou (2.5%; n = 120). In An. funestus s.l., Plasmodium falciparum sporozoite infection rate was estimated at 6.2% (n = 32) in Bloléquin, 8.7% (n = 23) in Odienné. No An. funestus s.l. specimens were found infected with P. falciparum sporozoite infection in Ouangolodougou and Aboisso. No P. falciparum sporozoite was detected in An. nili specimens in the four health districts. Among the local human populations, malaria incidence was higher in Odienné (39.7%; n = 45,376) and Bloléquin (37.6%; n = 150,205) compared to that in Ouangolodougou (18.3%; n = 131,629) and Aboisso (19.7%; n = 364,585).ConclusionAnopheles vector species diversity, abundance and Plasmodium sporozoite infection were high within the health districts along the borders of the country of Côte d’Ivoire, resulting in high malaria transmission among the local populations. Anopheles gambiae s.l. and An. funestus s.l. were found to be highly infected with Plasmodium in the health districts of Bloléquin and Odienné where higher malaria incidence was observed than the other districts. This study provides important information that can be used to guide Côte d’Ivoire National Malaria Control Programme for vector control decision-making, mainly in districts that are at the country borders.

A Systematic Review on Automatic Detection of Plasmodium Parasite

Plasmodium parasite is the main cause of malaria which has taken many lives. Some research works have been conducted to detect the Plasmodium parasite automatically. This research aims to identify the development of current research in the area of Plasmodium parasite detection. The research uses a systematic literature review (SLR) approach comprising three stages, namely planning, conducting, and reporting. The search process is based on the keywords which were determined in advance. The selection process involves the inclusion and exclusion criteria. The search yields 45 literatures from five different digital libraries. The identification process finds out that 28 methods are applied and mainly categorizes as machine learning algorithms with performance achievements between 60% and 95%. Overall, the research of Plasmodium parasite detection today has focused on the development with artificial intelligence specifically related to machine and deep learning. These approaches are believed as the most effective approach to detect Plasmodium parasites.

Comparison of capillary and venous blood for malaria detection using two PCR-based assays in febrile patients in Sierra Leone

BackgroundRapid and sensitive diagnostics are critical tools for clinical case management and public health control efforts. Both capillary and venous blood are currently used for malaria detection and while diagnostic technologies may not be equally sensitive with both materials, the published data on this subject are scarce and not conclusive.MethodsPaired clinical samples of venous and capillary blood from 141 febrile individuals in Bo, Sierra Leone, were obtained between January and May 2019 and tested for the presence of Plasmodium parasites using two multiplexed PCR assays: the FilmArray-based Global Fever Panel (GFP) and the TaqMan-based Malaria Multiplex Sample Ready (MMSR) assay.ResultsNo significant differences in Plasmodium parasite detection between capillary and venous blood for both assays were observed. The GFP assay was more sensitive than MMSR for all markers that could be compared (Plasmodium spp. and Plasmodium falciparum) in both venous and capillary blood.ConclusionsNo difference was found in malaria detection between venous and capillary blood using two different PCR-based detection assays. This data gives support for use of capillary blood, a material which can be obtained easier by less invasive methods, for PCR-based malaria diagnostics, independent of the platform.

A trial study on a mixture of Acridine and Giemsa stains and modified Quantitative Buffy Coat for detection of malaria parasites

Microscopy after Geimsa staining remains the gold standard technique for detection of malaria parasites while Acridine is a common fluorescent stain that enhances the visibility of parasites. Combination of both stains could further enhance the performance of microscopy technique. The aim of this study was to investigate the efficacy of staining with mixture of Acridine and Giemsa and modified Quantitative Buffy Coat technique in detection of plasmodium parasites. Five hundred and seventy (570) volunteered students attending the Kwara State University Health Center Malete with history malaria fever and tested positive for malaria parasite were re-examined with the two methods being investigated. Two millilitres of the blood sample from each subject was collected into EDTA container and mixed properly. Thick smears were made in duplicate from each sample and properly labeled, stained by Giemsa and Acridine-Giemsa techniques and examined under x100 objective of the microscope while the remaining blood samples were also tested by Modified QBC. The study assessed the Sensitivity, Specificity, Positive Predictive Value and Negative Predictive Value of both techniques with respect to Giemsa microscopy as a gold standard. Findings from the investigation showed that modified QBC and Acredine-Giemsa techniques recorded sensitivity of 100% and 99.7% respectively. Concentrated Acridine-Giemsa stain recorded higher specificity (90.7%) than modified QBC with 82.5% using Geimsa staining as a gold standard. The Positive Predictive Value (PPV) for Modified QBC and Acredine-Giemsa techniques were 96.8% and 96.5% respectively whereas the Negative Predictive Value (NPV) for both methods were 100% and 98.7% respectively. In conclusion, the finding depicts a keen competition between Acridine-Giemsa staining method and Modified QBC technique for detection of malaria parasites and there was no significant difference in the efficacy of both methods with respect to detection of malaria parasite.© 2019 International Formulae Group. All rights reservedKeywords: Acridine-Giemsa, Modified QBC, Efficacy, Microscopy, Gold standard

Effect of different habitat types on abundance and biting times of Anopheles balabacensis Baisas (Diptera: Culicidae) in Kudat district of Sabah, Malaysia

BackgroundWe investigated the effect of five common habitat types on the diversity and abundance of Anopheles spp. and on the biting rate and time of Anopheles balabacensis (currently the only known vector for Plasmodium knowlesi in Sabah) at Paradason village, Kudat, Sabah. The habitats were forest edge, playground area, longhouse, oil palm plantation and shrub-bushes area. Sampling of Anopheles was done monthly using the human landing catch method in all habitat types for 14 months (October 2013 to December 2014, excluding June 2014). The Anopheles species were morphologically identified and subjected to PCR assay for the detection of Plasmodium parasites. Generalised linear mixed models (GLMM) were applied to test the variation in abundance and biting rates of An. balabacensis in different habitat types.ResultsA total of 1599 Anopheles specimens were collected in the village, of which about 90% were An. balabacensis. Anopheles balabacensis was present throughout the year and was the dominant Anopheles species in all habitat types. The shrub bushes habitat had the highest Anopheles species diversity while forest edge had the greatest number of Anopheles individuals caught. GLMM analysis indicated that An. balabacensis abundance was not affected by the type of habitats, and it was more active during the early and late night compared to predawn and dawn. PCR assay showed that 1.61% of the tested An. balabacensis were positive for malaria parasites, most of which were caught in oil palm estates and infected with one to two Plasmodium species.ConclusionsThe identification of infected vectors in a range of habitats, including agricultural and farming areas, illustrates the potential for humans to be exposed to P. knowlesi outside forested areas. This finding contributes to a growing body of evidence implicating environmental changes due to deforestation, expansion of agricultural and farming areas, and development of human settlements near to forest fringes in the emergence of P. knowlesi in Sabah.

Investigations of dengue virus and <i>Plasmodium falciparum</i> among febrile patients receiving care at a tertiary health facility in Osogbo, south-west Nigeria

Both dengue and malaria are diseases of the tropical and sub-tropical regions having similar symptoms especially among the febrile subjects. A descriptive hospital-based study was carried out among randomly selected 170 consenting febrile patients ((m= 92; f=78) age ranged <1 to 55 years (mean age=27.2) attending LAUTECH teaching hospital, Osogbo, between March 2015 and February 2016. The detection of Plasmodium parasites was screened by RDT and confirmed by microscopy while primary dengue virus detection was investigated using ELISA technique. The overall rate of 41.7% was obtained for P. falciparum while 1.8% was reported for dengue infection. Analysis by age and sex showed that the rate of P. falciparum was higher among male (42.0%) than female with highest rate (22.2%) was peaked among age range 16-25 years and 36-45 years respectively with no statistical associations (p = 0.469; OR =1.26, 95% CI 1.21-2.31). Infection rate for dengue was higher in females (2.6%) than males (1.2%) and found among age ranged of 16-25 and 26-35 years respectively. Also, dengue virus P. falciparum co-infection rate (0.6%) was found among participant age ranged 26-35 years. However, there were no statistical associations between age/sex in relation to dengue and malaria infections (p = 0.359; OR = 1.16, 95% CI 1.34-2.66) in this study. This study reported high rates of dengue and malaria infections among the community dwellers. There is need to ascertain the actual seroprevalence of dengue and malaria infections among the febrile patients in rural and urban centers together with those accessing care in our healthcare facilities in Nigeria.Keywords: Dengue/malaria infections; mosquitoes; fever; Osogbo community; Nigeria

Loop-mediated isothermal amplification (LAMP) and Polymerase Chain Reaction (PCR) as quality assurance tools for Rapid Diagnostic Test (RDT) malaria diagnosis in Northern Namibia.

Malaria cases sometimes go undetected using RDTs due to their inaccurate use, poor storage conditions and failure to detect low parasitaemia (<50parasites/μL). This could result in continuous transmission of malaria and sustenance of parasite reservoirs. Molecular diagnostic tools are more sensitive and specific than RDTs in the detection of plasmodium parasites. However, the Polymerase Chain Reaction (PCR) is not routinely used because equipment and reagents are expensive and requires highly skilled personnel. Loop-mediated isothermal amplification (LAMP) is a relatively new molecular diagnostic tool for malaria with all the advantages of PCR (sensitive and specific) without the mentioned disadvantages. However, it has not been evaluated extensively as a point of care diagnostic in the field. One hundred and fifteen used RDTs were collected from health facilities in Northern Namibia in a blind study and PCR and LAMP were used to determine the presence of Plasmodium DNA. The sensitivities and PPV were 40.91% and 90% respectively for RDTs, 72.73% and 100% respectively for PCR with LAMP as the golden standard. In low malaria transmission settings, LAMP can be also be considered for use as a surveillance tool to detect all sources of malaria and determine proportion of low parasitaemia infections in order to eliminate them.

Does it take three to tango? An unsuspected multimorbidity of CD8+ T cell lymphoproliferative disorder, malaria, and EBV infection

BackgroundMalaria is known to cause acute and deadly complications. However, malaria can cause unforeseen pathologies due to its chronicity. It increases the risk of endemic Burkitt Lymphoma development by inducing DNA damage in germinal centre (GC) B cells, and leading higher frequency of Epstein–Barr virus (EBV)-infected cells in GCs. EBV is well known for its tropism for B cells. However, less is known about EBV’s interaction with T cells and its association with T cell lymphoma.Case presentationA 43-year-old Sudanese male admitted to hospital in Istanbul, Turkey, a non-endemic country, with hyperpigmented painful skin rashes on his whole body. A complete blood count and a peripheral blood smear during admission revealed large granular lymphocytes (LGLs) with abnormally higher CD8 T cell numbers. Additional skin biopsy and pathology results were compatible with CD8+ T cell lymphoproliferative disorder with skin involvement. Patient was treated and discharged. However, a pathologist noticed unusual structures in skin tissue samples. Careful evaluation of skin biopsy samples by polarized microscopy revealed birefringent crystalloid structures resembling malarial haemozoin mainly loaded in macrophages and giant histiocytes. After purification of DNA from the skin biopsy samples, nested PCR was performed for the detection of Plasmodium parasites and Plasmodium falciparum DNA was amplified. Because, the co-presence of EBV infection with malaria is a well-known aetiology of lymphoma, EBV-early RNA (EBER) transcripts were investigated in paraffin-embedded tissue samples and found to be positive in macrophage-like histiocytes.ConclusionsThis is a unique case of malaria and EBV infection in a T-LGL lymphoma patient who presented in a non-endemic country. This case emphasizes the clinical importance of EBV monitoring in T-LGL patients with skin involvement. Notably, Plasmodium infection should be examined in patients from malaria endemic regions by pathological and molecular investigations.

Studies on asymptomatic malaria, prevention and treatment seeking behaviours in Abeokuta, south-west Nigeria

Recurrent cycles of fevers and chills are obvious symptoms for malaria disease usually necessitating treatment. However, some Plasmodium infections could be without symptoms, leading to late diagnosis and delayed treatment- seeking behaviours. The objective of this study was to determine the prevalence of asymptomatic malaria infections, and document associated prevention and treatment-seeking behaviours among literates in Abeokuta. A cross-sectional study design involving 309 attendees at the 2016 World Malaria Day Celebration event held at the Federal University of Agriculture, Abeokuta was conducted. Finger-prick blood samples were collected for Plasmodium parasite detection by malaria rapid diagnostic test (mRDT) and anthropometric indices such as weight, height and age were also collected to estimate Body-Mass-Index. An already pre-tested questionnaire was also used to collect information on malaria prevention and treatment-seeking behaviours. Data were analysed in SPSS 20.0 software. Results revealed that the prevalence of asymptomatic malaria was 7.4% (23/309), with no significant association between sex, febrile illness and Body Mass Index ( p >0.05). Participants aged 15-25 years were more infected with malaria than other age groups. Only 58.3% of the participants have heard of Long Lasting Insecticidal Nets (LLINs) and about 78% do not have mosquito bed-net. Self-diagnosis for the disease was more common (60.8%) among the participants, compared to other measures; seeking laboratory test (26.5%) and clinical diagnosis (9.1%). A good proportion of the participants (73.1%) rely on Artemisinin-based Combination Therapy (ACT) drugs for treating malaria, although few others still rely on non-ACT drugs (17.8%), chloroquine (1.9%) and herbs (1.6%). Findings show existence of asymptomatic malaria. Also, access to and utilization of malaria prevention and control commodities is poor among the literates. There is thus a need to intensify efforts in addressing these concerns as we move towards eliminating the disease. Keywords: Asymptomatic malaria; World Malaria Day; prevalence; Abeokuta; Nigeria

MALDI-TOF MS as an innovative tool for detection of Plasmodium parasites in Anopheles mosquitoes

BackgroundMalaria is still a major public health issue worldwide, and one of the best approaches to fight the disease remains vector control. The current methods for mosquito identification include morphological methods that are generally time-consuming and require expertise, and molecular methods that require laboratory facilities with relatively expensive running costs. Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) technology, routinely used for bacterial identification, has recently emerged in the field of entomology. The aim of the present study was to assess whether MALDI-TOF MS could successfully distinguish Anopheles stephensi mosquitoes according to their Plasmodium infection status.MethodsC57BL/6 mice experimentally infected with Plasmodium berghei were exposed to An. stephensi bites. For the determination of An. stephensi infection status, mosquito cephalothoraxes were dissected and submitted to mass spectrometry analyses and DNA amplification for molecular analysis. Spectra were grouped according to mosquitoes’ infection status and spectra quality was validated based on intensity and reproducibility within each group. The in-lab MALDI-TOF MS arthropod reference spectra database, upgraded with representative spectra from both groups (infected/non-infected), was subsequently queried blindly with cephalothorax spectra from specimens of both groups.ResultsThe MALDI TOF MS profiles generated from protein extracts prepared from the cephalothorax of An. stephensi allowed distinction between infected and uninfected mosquitoes. Correct classification was obtained in blind test analysis for (79/80) 98.75% of all mosquitoes tested. Only one of 80 specimens, an infected mosquito, was misclassified in the blind test analysis.ConclusionsMatrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry appears to be a promising, rapid and reliable tool for the epidemiological surveillance of Anopheles vectors, including their identification and their infection status.

Prevalence of asymptomatic malaria infection and use of different malaria control measures among primary school children in Morogoro Municipality, Tanzania.

BackgroundMalaria is a public health problem in Tanzania affecting all age groups. It is known that school children are the age group most commonly infected with malaria parasites. Their infections are usually asymptomatic, go unnoticed and thus never get treated, result in anaemia, reduced ability to concentrate and learn in school and if fallen sick may lead to school absenteeism. Effective malaria control requires frequent evaluation of effectiveness of different malaria interventions.MethodsA cross-sectional study design involving 317 out of 350 school children aged 6–13 years from five primary schools within municipality was conducted. Multistage cluster sampling and simple random sampling methods were used to obtain primary school and study participants, respectively. Finger-prick blood samples were collected for Plasmodium parasite detection by malaria rapid diagnostic test (mRDT) and haemoglobin level assessment by Easy Touch® GHb system machine. A questionnaire was administered to assess use of insecticide-treated nets (ITNs) and anti-malarial drugs.ResultsThe prevalence of asymptomatic malaria was 5.4 % (95 % CI 3.3–8.6 %) and anaemia was 10.1 % (95 % CI 7.2–13.9 %). School children aged 6–9 years were more affected by malaria than those aged 10–13 years. The proportion of ITNs used was 90.6 % (95 % CI 86.3–93.9 %) while that of artemisinin combination therapy (ACT) was 71.9 % (95 % CI 66.2–77.1 %).ConclusionFindings show existence of asymptomatic malaria and walking anaemia among primary school children in Morogoro municipality. The majority of school children reported use of ITNs and ACT for malaria control. These findings provide a rationale for using schools and school children to assess effectiveness of malaria interventions.

Detection and Species Identification of Malaria Parasites by Isothermal tHDA Amplification Directly from Human Blood without Sample Preparation

We report the clinical and analytical performance of an isothermal thermophilic helicase-dependent amplification assay for blood Plasmodium parasite detection and species-level identification. The assay amplifies the 18S rRNA gene fragment of all Plasmodium species and uses a species-specific probe and a pan-malarial probe to definitively identify Plasmodium falciparum from other infectious Plasmodium species. Amplicon-probe hybridization products are detected with a disposable dipstick enclosed in a cassette. With a pan-malarial-positive and P. falciparum-negative result, an additional test is performed to detect if the pan-malarial-positive band was the result of the presence of Plasmodium vivax. The assay uses only 2 μL of human whole blood directly for a 50-μL amplification reaction, without any pre-amplification processing. The clinical performance of the assay was validated using 88 samples from New York patients suspected of malaria or babesiosis. The overall sensitivity of the assay was 96.6% (95% CI, 87.3% to 99.4%), and the specificity was 100% (95% CI, 85.4% to 100%), compared with gold standard microscopy and a laboratory-developed molecular assay, respectively. The analytical sensitivity was 50 copies of DNA per assay or 200 parasites per microliter of blood, and the assay can detect samples with parasitemia levels <1%. This novel molecular diagnostic assay requires minimal laboratory instrumentation and uses un-processed blood as input; it can be readily performed in the field.

Detection of plasmodium parasites from images of thin blood smears

Malaria is the leading cause of morbidity and mortality in tropical and subtropical countries. Conventional microscopy is the Gold standard in the diagnosis of the disease. However, it is prone to some shortcomings which include time consumption and difficultness in reproducing results. Alternative diagnosis techniques which yield superior results are quite expensive and hence inaccessible to developing countries where the disease is prevalent. Thus in this work, an accurate, speedy and affordable system of malaria detection using stained thin blood smear images was developed. The method uses Artificial Neural Network (ANN) to test for the presence of plasmodium parasites in thin blood smear images. Images of infected and non-infected erythrocytes were acquired, pre-processed, relevant features extracted from them and eventually diagnosis was made based on the features extracted from the images. Diagnosis entailed detection of plasmodium parasites. Classification accuracy of 95.0% in detection of infected erythrocyte was achieved with respect to results obtained by expert microscopists. The study revealed that artificial neural network (ANN) classifiers trained with colour features of infected stained thin blood smear images are suitable for detection. It was further shown that ANN classifiers can be trained to perform image segmentation.

Droplet Microfluidics Platform for Highly Sensitive and Quantitative Detection of Malaria-Causing Plasmodium Parasites Based on Enzyme Activity Measurement

We present an attractive new system for the specific and sensitive detection of the malaria-causing Plasmodium parasites. The system relies on isothermal conversion of single DNA cleavage-ligation events catalyzed specifically by the Plasmodium enzyme topoisomerase I to micrometer-sized products detectable at the single-molecule level. Combined with a droplet microfluidics lab-on-a-chip platform, this design allowed for sensitive, specific, and quantitative detection of all human-malaria-causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite/μL. Moreover, the setup allowed for detection of Plasmodium parasites in noninvasive saliva samples from infected patients. During recent years malaria transmission has declined worldwide, and with this the number of patients with low-parasite density has increased. Consequently, the need for accurate detection of even a few parasites is becoming increasingly important for the continued combat against the disease. We believe that the presented droplet microfluidics platform, which has a high potential for adaptation to point-of-care setups suitable for low-resource settings, may contribute significantly to meet this demand. Moreover, potential future adaptation of the presented setup for the detection of other microorganisms may form the basis for the development of a more generic platform for diagnosis, fresh water or food quality control, or other purposes within applied or basic science.