Parasite DNA Research Articles

First molecular data on the human roundworm Ascaris lumbricoides species complex from the Bronze and Iron Age in Hallstatt, Austria

Palaeoparasitological studies can provide valuable information on the emergence, distribution, and elimination of parasites during a particular time in the past. In the prehistoric salt mines of Hallstatt, located in the Austrian Alps, human faeces have been conserved in salt. The aim of this study was to recover ancient DNA of intestinal parasites from these coprolites. Altogether, 35 coprolites from the Hallstatt salt mines, dating back to the Bronze Age mining phase (1158–1063 BCE) and the Iron Age mining phase (750–662 BCE), respectively, were analysed by microscopy and molecular methods. In 91% of the coprolite samples, eggs of soil-transmitted helminths (STH), namely of Trichuris and/or Ascaris were detected by light microscopy. The Ascaris eggs were exceptionally well preserved. For further analysis, DNA was extracted from the palaeofaecal samples and species-specific primers targeting different genes were designed. While amplification of Trichuris DNA remained unsuccessful, sequence data of A. lumbricoides species complex were successfully obtained from 16 coprolites from three different genes, the mitochondrial cytochrome c oxidase subunit 1 gene (cox1), the mitochondrial cytochrome B gene (cytB) and the mitochondrial NADH dehydrogenase subunit 1 gene (nadh1). Importantly, these included two Ascaris sequences from a coprolite from the Bronze Age, which to the best of our knowledge are the first molecular data of this genus from this period.

Genome-wide binding sites of Plasmodium falciparum mini chromosome maintenance protein MCM6 show new insights into parasite DNA replication

Multiple rounds of DNA replication take place in various stages of the life cycle in the human malaria parasite Plasmodium falciparum. Previous bioinformatics analysis has shown the presence of putative Autonomously Replicating Sequence (ARS) like sequences in the Plasmodium genome. However, the actual sites and frequency of replication origins in the P. falciparum genome based on experimental data still remain elusive. Minichromosome maintenance (MCM) proteins are recruited by the Origin recognition complex (ORC) to the origins of replication in eukaryotes including P. falciparum. We used PfMCM6 for chromatin immunoprecipitation followed by sequencing (ChIP-seq) in the quest for identification of putative replication origins in the parasite. PfMCM6 DNA binding sites annotation revealed high enrichment at exon regions. This is contrary to higher eukaryotes that show an inclination of origin sites towards transcriptional start sites. ChIP-seq results were further validated by ChIP-qPCR results as well as nascent strand abundance assay at the selected PfMCM6 enriched sites that also showed preferential binding of PfORC1 suggesting potential of these sites as origin sites. Further, PfMCM6 ChIP-seq data showed a positive correlation with previously published histone H4K8Ac genome-wide binding sites but not with H3K9Ac sites suggesting epigenetic control of replication initiation sites in the parasites. Overall, our data show the genome-wide distribution of PfMCM6 binding sites with their potential as replication origins in this deadly human pathogen that not only broadens our knowledge of parasite DNA replication and its unique biology, it may help to find new avenues for intervention processes.

Crosstalk between RNA m6A and DNA methylation regulates transposable element chromatin activation and cell fate in human pluripotent stem cells.

Transposable elements (TEs) are parasitic DNA sequences accounting for over half of the human genome. Tight control of the repression and activation states of TEs is critical for genome integrity, development, immunity and diseases, including cancer. However, precisely how this regulation is achieved remains unclear. Here we develop a targeted proteomic proximity labeling approach to capture TE-associated proteins in human embryonic stem cells (hESCs). We find that the RNA N6-methyladenosine (m6A) reader, YTHDC2, occupies genomic loci of the primate-specific TE, LTR7/HERV-H, specifically through its interaction with m6A-modified HERV-H RNAs. Unexpectedly, YTHDC2 recruits the DNA 5-methylcytosine (5mC)-demethylase, TET1, to remove 5mC from LTR7/HERV-H and prevent epigenetic silencing. Functionally, the YTHDC2/LTR7 axis inhibits neural differentiation of hESCs. Our results reveal both an underappreciated crosstalk between RNA m6A and DNA 5mC, the most abundant regulatory modifications of RNA and DNA in eukaryotes, and the fact that in hESCs this interplay controls TE activity and cell fate.

Molecular epidemiology of non-falciparum Plasmodium infections in three different areas of the Ivory Coast

BackgroundMalaria is a major public health problem, particularly in the tropical regions of America, Africa and Asia. Plasmodium falciparum is not only the most widespread but also the most deadly species. The share of Plasmodium infections caused by the other species (Plasmodium ovale and Plasmodium malariae) is clearly underestimated. The objective of the study was to determine the molecular epidemiology of plasmodial infection due to P. malariae and P. ovale in Côte d'Ivoire.MethodsThe study was cross-sectional. The study participants were recruited from Abengourou, San Pedro and Grand-Bassam. Sample collection took place from May 2015 to April 2016. Questionnaires were administered and filter paper blood samples were collected for parasite DNA extraction. The molecular analysis was carried out from February to March 2021. A nested PCR was used for species diagnosis. The data was presented in frequencies and proportions.ResultsA total of 360 patients were recruited, including 179 men (49,7%) for 181 women (50,3%). The overall Plasmodium positive rate was 72.5% (261/360). The specific index was 77.4% and 1.5% for P. falciparum and P. malariae in mono-infection, respectively. There was also 15% P. falciparum and P. malariae co-infection, 3.4% P. falciparum and P. ovale co-infection and 2.3% P. falciparum, P. malariae and P. ovale triple-infection. Typing of P. ovale subspecies showed a significant predominance of P. ovale curtisi (81.2% of cases).ConclusionPlasmodium falciparum remains the most prevalent malaria species in Côte d'Ivoire, but P. malariae and P. ovale are also endemic mostly in co-infection. Malaria elimination requires a better understanding of the specific epidemiological characteristics of P. malariae and P. ovale with a particular emphasis on the identification of asymptomatic carriers.

Genetic Diversity of Toxoplasma gondii by Serological and Molecular Analyzes in Different Sheep and Goat Tissues in Northeastern Iran.

Toxoplasmosis is a parasitic disease caused by compilation protozoan agent Toxoplasma gondii, leading to significant financial and quality-adjusted life-year losses. Overcooked or raw meat consumption has been a considerable transmission route. The present study was conducted to determine the seropositivity rate of T. gondii in sheep and goats by serological and molecular tests and genotyping of obtained isolates in northeast Iran. Blood and tissue samples (diaphragm, heart) of 296 animals (including 168 sheep and 128 goats) were collected from the slaughterhouse in Quchan Country from august 2016 to April 2017. Modified agglutination test (MAT) and the PCR method performed to detect parasite DNA on tissues.PCR-RFLP method of GRA6 gene was used to determine the genotype of T. gondii. In addition, sequencing analysis was performed to evaluate the Toxoplasma type strains. Serum positive for MAT results were found in 27.4% of sheep and 23.4% of goats. Positive PCR of B1 gene results in diaphragm and heart tissues of sheep and goats was 47.8% and 26.1%, 40% and 23.3%, respectively. PCR of GRA6 gene results were positive in 10 samples that RFLP technique results using MseI enzyme revealed genotype I. Sequencing and phylogenetic analysis revealed DNA of all samples was closely related to Toxoplasma type I. Concerning the high seropositivity rate of toxoplasmosis, undertaking an appropriate preventive program for reducing the prevalence of T. gondii infection by raw or undercooked meat consumption of livestock is recommended. Our study supports the notion that these animals' consumption of raw and undercooked meat can be a probable source of human toxoplasmosis.

Design of a Protocol for Soil-Transmitted Helminths (in Light of the Nematode Toxocara canis) DNA Extraction from Feces by Combining Commercially Available Solutions.

One of the main challenges for the mass introduction of the molecular diagnostics of soil-transmitted helminths (STHs) into clinical practice is the lack of a generally recognized effective method for isolating parasitic DNA from fecal samples. In the present study, we assessed the effects of various pretreatment procedures on the efficiency of removing PCR inhibitors and extracting Toxocara canis DNA from feces. We evaluated the effectiveness of four destructive methods (bead beating, the action of temperature-dependent enzymes, freeze-heat cycles, and incubation in a lysis buffer) on the integrity of T. canis eggs and the efficiency of DNA extraction. Also, we evaluated the effects of prewashes and the use of commercial concentrators on DNA extraction from fecal samples contaminated with T. canis eggs. A bead beating procedure was sufficient to destroy the T. canis eggs, while the effects of enzymes and freeze-heat cycles did not lead to a significant destruction of the eggs or the release of Toxocara DNA. Helminth DNA isolation protocols that do not include a bead beating step are not preferred. The preconcentration of STH eggs from feces using a commercial concentrator and subsequent washing can significantly increase the yield of DNA from STHs and reduce PCR inhibition.

Amplicon sequencing allows differential quantification of closely related parasite species: an example from rodent Coccidia (Eimeria)

BackgroundQuantifying infection intensity is a common goal in parasitological studies. We have previously shown that the amount of parasite DNA in faecal samples can be a biologically meaningful measure of infection intensity, even if it does not agree well with complementary counts of transmission stages (oocysts in the case of Coccidia). Parasite DNA can be quantified at relatively high throughput using quantitative polymerase chain reaction (qPCR), but amplification needs a high specificity and does not simultaneously distinguish between parasite species. Counting of amplified sequence variants (ASVs) from high-throughput marker gene sequencing using a relatively universal primer pair has the potential to distinguish between closely related co-infecting taxa and to uncover the community diversity, thus being both more specific and more open-ended.MethodsWe here compare qPCR to the sequencing-based amplification using standard PCR and a microfluidics-based PCR to quantify the unicellular parasite Eimeria in experimentally infected mice. We use multiple amplicons to differentially quantify Eimeria spp. in a natural house mouse population.ResultsWe show that sequencing-based quantification has high accuracy. Using a combination of phylogenetic analysis and the co-occurrence network, we distinguish three Eimeria species in naturally infected mice based on multiple marker regions and genes. We investigate geographical and host-related effects on Eimeria spp. community composition and find, as expected, prevalence to be largely explained by sampling locality (farm). Controlling for this effect, the novel approach allowed us to find body condition of mice to be negatively associated with Eimeria spp. abundance.ConclusionsWe conclude that amplicon sequencing provides the underused potential for species distinction and simultaneous quantification of parasites in faecal material. The method allowed us to detect a negative effect of Eimeria infection on the body condition of mice in the natural environment.Graphical

One piece of the puzzle: Modeling vector presence and environment reveals seasonality, distribution, and prevalence of sandflies and Leishmania in an expansion area

The recent geographic spread of Leishmania infantum along the borders of Argentina, Brazil and Paraguay has been highlighted. In our previous study, Lutzomyia longipalpis was found in 55 of 123 patches surveyed, and in some patches, sandflies were found at higher densities, forming hotspots. Based on the One Health approach, we investigated the seasonality of the vector, the presence of parasite DNA, and the environmental factors that contribute to vector and parasite dispersal in these previously described hotspots in Foz do Iguaçu, Brazil. Entomological surveys were conducted monthly for one year. Fourteen hotspots peridomicile and six intradomicile were sampled. PCR was used to assess the prevalence of Leishmania DNA in sandflies. Zero-inflated negative binomial regression was used to determine the association of micro- and mesoscale environmental variables with the occurrence and abundance of the three most abundant sandfly species sampled. A total of 3543 species were captured, with Lutzomyia longipalpis being the predominant species (71.78%) of the 13 species found. Evandromyia edwardsi, Expapillata firmatoi, Micropygomyia ferreirana and Pintomyia christenseni were reported for the first time in the region. NDVI, distance to water, precipitation, west-to-east wind, wind speed, maximum and minimum relative humidity, and sex were significant variables associated with vector presence/abundance in the environment. Vector presence/abundance in the peridomicile was associated with precipitation, altitude, maximum temperature, minimum and maximum relative humidity, west-to-east wind, wind speed, and sex. Leishmania DNA was detected in an average of 21% of Lu. longipalpis throughout the year. Vector abundance is concentrated in urban and peri-urban areas, with some specimens present in different parts of the city and some sites with high vector abundance. This distribution suggests that the risk of actual contact between humans and parasite vectors in urban areas during the epidemic period is associated with patches of peri-urban vegetation and then extends into urban areas.

A parasite DNA binding protein with potential to influence disease susceptibility acts as an analogue of mammalian HMGA transcription factors.

Intracellular pathogens construct their environmental niche, and influence disease susceptibility, by deploying factors that manipulate infected host cell gene expression. Theileria annulata is an important tick-borne parasite of cattle that causes tropical theileriosis. Excellent candidates for modulating host cell gene expression are DNA binding proteins bearing AT-hook motifs encoded within the TashAT gene cluster of the parasite genome. In this study, TashAT2 was transfected into bovine BoMac cells to generate three expressing and three non-expressing (opposite orientation) cell lines. RNA-Seq was conducted and differentially expressed (DE) genes identified. The resulting dataset was compared with genes differentially expressed between infected cells and non-infected cells, and DE genes between infected cell lines from susceptible Holstein vs tolerant Sahiwal cattle. Over 800 bovine genes displayed differential expression associated with TashAT2, 209 of which were also modulated by parasite infection. Network analysis showed enrichment of DE genes in pathways associated with cellular adhesion, oncogenesis and developmental regulation by mammalian AT-hook bearing high mobility group A (HMGA) proteins. Overlap of TashAT2 DE genes with Sahiwal vs Holstein DE genes revealed that a significant number of shared genes were associated with disease susceptibility. Altered protein levels encoded by one of these genes (GULP1) was strongly linked to expression of TashAT2 in BoMac cells and was demonstrated to be higher in infected Holstein leucocytes compared to Sahiwal. We conclude that TashAT2 operates as an HMGA analogue to differentially mould the epigenome of the infected cell and influence disease susceptibility.

Anti-malarial resistance in Mozambique: Absence of Plasmodium falciparum Kelch 13 (K13) propeller domain polymorphisms associated with resistance to artemisinins

BackgroundMalaria remains one of the most serious public health problems in sub-Saharan Africa and Mozambique is the world's fourth largest contributor, with 4.7% of disease cases and 3.6% of total deaths due to malaria. Its control relies on the fight against the vector and treatment of confirmed cases with anti-malarial drugs. Molecular surveillance is an important tool for monitoring the spread of anti-malarial drug resistance.MethodsA cross-sectional study recruited 450 participants with malaria infection detected by Rapid Diagnostic Tests, from three different study sites (Niassa, Manica and Maputo) between April and August 2021. Correspondent blood samples were collected on filter paper (Whatman® FTA® cards), parasite DNA extracted and pfk13 gene sequenced using Sanger method. SIFT software (Sorting Intolerant From Tolerant) was used, predict whether an amino acid substitution affects protein function.ResultsNo pfkelch13-mediated artemisinin resistance gene mutation was detected in this study settings. However, non-synonymous mutations were detected at prevalence of 10.2%, 6% and 5% in Niassa, Manica and Maputo, respectively. Most (56.3%) of the reported non-synonymous mutations were due to substitution at the first base of the codon, 25% at the second base and 18.8% at the third base. Additionally, 50% of non-synonymous mutations showed a SIFTscore bellow cut off value of 0.05, therefore, they were predicted to be deleterious.ConclusionThese results do not show an emergence of artemisinin resistance cases in Mozambique. However, the increased number of novel non-synonymous mutations highlights the relevance of increasing the number of studies focused on the molecular surveillance of artemisinin resistance markers, for its early detection.

Foodborne protozoan parasites in fresh mussels and oysters purchased at retail in Canada

Studies worldwide have reported the presence of protozoan parasites in a variety of commercial bivalve shellfish. The uptake of these parasites by shellfish occurs during filter feeding in faecally-contaminated waters. The objective of the present study was to determine the prevalence of Giardia, Cryptosporidium and Toxoplasma in fresh, live shellfish purchased in three Canadian provinces as part of the retail surveillance activities led by FoodNet Canada (Public Health Agency of Canada). Packages containing mussels (n = 253) or oysters (n = 130) were purchased at grocery stores in FoodNet Canada sentinel sites on a biweekly basis throughout 2018 and 2019, and shipped in coolers to Health Canada for testing. A small number of packages were not tested due to insufficient quantity or poor quality. Following DNA extraction from homogenized, pooled tissues, nested PCR and DNA sequencing were used to detect parasite-specific sequences. Epifluorescence microscopy was used to confirm the presence of intact cysts and oocysts in sequence-confirmed PCR-positive samples. Giardia duodenalis DNA was present in 2.4 % of 247 packages of mussels and 4.0 % of 125 packages of oysters, while Cryptosporidium parvum DNA was present in 5.3 % of 247 packages of mussels and 7.2 % of 125 packages of oysters. Toxoplasma gondii DNA was only found in mussels in 2018 (1.6 % of 249 packages). Parasite DNA was detected in shellfish purchased in all three Canadian provinces sampled, and there was no apparent seasonal variation in prevalence. While the present study did not test for viability, parasites are known to survive for long periods in the marine environment, and these findings suggest that there is a risk of infection, especially when shellfish are consumed raw.

Demodex carolliae in a colony of Seba's short-tailed bats (Carollia perspicillata): clinical, pathological and parasitological findings

Seba's short-tailed bats (Carollia perspicillata) are a frugivorous species native to Central and South America. Despite their importance as a reservoir for zoonotic pathogens and their popularity in zoological collection and as research models, there are relatively few reports on non-zoonotic diseases of bats. Mites of the genus Demodex are obligate commensals of the skin of a range of mammals, are highly host-specific and are not associated with clinical disease when present in low numbers. However, infestation with high numbers can result in severe or even fatal disease and substantially affect the well-being of the animals. The clinical, pathological and parasitological findings in 12 Seba's short-tailed bats with demodicosis from a colony kept at Munich Zoo Hellabrunn between 1992 and 2021 are described in this report. From 2002, skin lesions became apparent on the head, especially the periocular region, nose and ears, as well as the genital area of some animals. In advanced cases, skin changes were also present on the abdomen, back and extremities. Gross findings typically included alopecia and thickening of the skin, with the formation of papules, reflecting cystically dilated hair follicles containing myriads of demodecid mites. Histologically, lesions were characterized by a paucicellular lymphocytic dermatitis and folliculitis with perifollicular fibrosis, epidermal hyperplasia, orthokeratotic hyperkeratosis and disproportionately high numbers of intrafollicular arthropods. Demodex carolliae was identified morphologically by light, phase-contrast and electron microscopy. Further characterization was achieved by extraction of parasitic DNA and partial gene sequencing of two mitochondrial genes, 16S rDNA and cox1. This is the first clinicopathological description of generalized demodicosis in Seba's short-tailed bats and includes the first molecular characterization of D. carolliae with provision of a GenBank entry.

Molecular Screening of Echinococcus spp. and Other Cestodes in Wild Carnivores from Central Italy.

Tapeworm infections are among the most relevant parasitic diseases in humans and animals. Tapeworms from the Genus Echinococcus are particularly important as they can cause cystic or alveolar echinococcosis. A molecular screening was performed on 279 fecal samples collected from carcasses of wild carnivores from Central Italy using PCR targeting diagnostic fragments of nad1, rrnS, and nad5 genes. Samples positive for either Taenia spp. or Echinococcus granulosus were sequenced to taxonomically identify the parasitic DNA. Of the 279 samples, 134 (48.0%) gave positive results in the multiplex PCR. Only one (0.4%) sample from an Apennine wolf tested positive for Echinococcus granulosus sensu stricto (genotype G3), whereas no sample tested positive for E. multilocularis. The most frequently detected tapeworms were: Mesocestoides corti (syn M. vogae) (12.9%), M. litteratus (10.8%), Taenia serialis (9.3%), and T. hydatigena (6.5%), other tapeworms were rarely detected. The results suggest that Echinococcus infections in Central Italy do not seem to be sustained by sylvatic cycles, confirming the absence of E. multilocularis in Central Italy. The survey corroborates, yet again, the importance of passive surveillance of wild animals that can serve as reservoirs for zoonotic pathogens, especially on wild canids that in other areas are strongly implicated in the transmission of E. granulosus and E. multilocularis.

First evidence for development of Plasmodium relictum (Grassi and Feletti, 1891) sporozoites in the salivary glands of Culex modestus Ficalbi, 1889.

The competence of insect vectors to transmit diseases plays a key role in host-parasite interactions and in the dynamics of avian malaria and other haemosporidian infections (Apicomplexa, Haemosporida). However, the presence of parasite DNA in the body of blood-sucking insects does not always constitute evidence for their competence as vectors. In this study, we investigate the susceptibility of wild-caught mosquitoes (Culex spp.) to complete sporogony of Plasmodium relictum (cyt b lineage SGS1) isolated from great tits (Parus major L., 1758). Adult female mosquitoes were collected with a CO2 bait trap overnight. A set of 50 mosquitoes was allowed to feed for 3 h at night on a single great tit infected with P. relictum. This trial was repeated on 6 different birds. The bloodfed mosquitoes that survived (n = 68) were dissected within 1-2 days (for ookinetes, n = 10) and 10-33 days post infection (for oocysts and sporozoites, n = 58) in order to confirm the respective parasite stages in their organs. The experiment confirmed the successful development of P. relictum (cyt b lineage SGS1) to the stage of sporozoites in Culex pipiens L., 1758 (n = 27) and in Culex modestus (n = 2). Our study provides the first evidence that C. modestus is a competent vector of P. relictum isolated from great tits, suggesting that this mosquito species could also play a role in the natural transmission of avian malaria.

Malignant human thyroid neoplasms associated with blood parasitic (haemosporidian) infection

A retrospective investigation of archival cytology specimens obtained and verified by a fine-needle aspiration biopsy from patients with follicular, papillary, and medullary human thyroid cancers revealed haemosporidian (blood parasitic) infection in thyrocytes (schizogony) and erythrocytes. The exoerythrocytic stage of infection is represented by finding microschizonts. Cytologic material was stained with RomanowskyGiemsa dye in medical laboratories. Original specimens were stained (re-stained) with Schiff reagent according to the Feulgen method to clarify location of thyrocyte DNA and hemosporidian pathogens, wherein fuchsine was incorporated into DNA molecules after they were hydrolyzed by hydrochloric acid to stain specimens into red-violet color. An intentionally unstained hemosporidian protoplasm during blood parasitic infection was observed as a light band around erythrocyte nuclei. In follicular thyroid cancer, thyrocyte Feulgen staining revealed nuclear DNA and parasitic DNA (haemosporidium nuclei) as punctate inclusions and rings diffusely distributed in the thyrocyte cytoplasm. The thyrocyte cytoplasm and nuclei were vacuolated, with thyrocyte nuclei being deformed, flattened, and displaced to the cell periphery. The erythrocytes contained haemosporidian nuclei (DNA). In papillary thyroid cancer, we were able to localize the nuclear DNA of thyrocytes and the parasitic DNA as punctate inclusions diffusely distributed in the thyrocyte cytoplasm. Two or more polymorphic nuclei may be positioned eccentrically in the hyperplastic cytoplasm. Haemosporidian microschizonts were found circumnuclearly in thyrocytes as well as an exoerythrocytic stage in the blood. In medullary thyroid cancer, the hyperplastic cytoplasm of thyrocytes contained eccentrically located nuclei (DNA) of thyrocytes and small haemosporidian nuclei (DNA), which may occupy the whole thyrocyte. There were thyrocytes with vacuolated cytoplasm and prominent nuclear polymorphism. The size of hyperplastic nuclei was several times larger than that of normal thyrocyte nuclei. The color of stained thyrocyte cytoplasmic and nuclear vacuoles was less red-violet compared with that of surrounding tissues, which potentially indicates the presence of parasitic DNA inside them. The intra-erythrocyte nuclear haemosporidian material of varying sizes in papillary and medullary cancers may evidence about various species and/or pathogen generation. Intracellular parasitism of haemosporidian infection in thyrocytes (schizogony) associated with three thyroid cancer types leads to marked thyrocyte cytoplasmic hyperplasia, cytoplasmic vacuolization, and nuclear vacuolization. Multinucleated thyrocytes with incomplete cytokinesis emerge. Nuclear deformation occurs, which leads to decreased nucleus size, flattening and displacement to the cell periphery, with high risk of DNA mutations and deletions in affected cells, reaching a neoplastic level.

Survey in Iranian Malaria Blood Donation Deferral: Laboratory Evaluation in Malaria Blood Donation Deferral in the Iranian Blood Centers

Introduction: Transfusion-transmitted malaria (TTM), from a malaria infected donor, is an accidental Plasmodium infection, which is one of the concerns of blood transfusion issue in the world. Countries have adopted different strategies for donor selection and improving criteria to overcome these concerns, In the Iranian Blood Transfusion Organization (IBTO), donors who refer to blood transfusion centers after registration are screened by the physicians based on interview and examination. This prevention due to the possibility of malaria infection is called malaria donor deferral strategy.
 Methods: In this study 248 malaria blood donation deferral samples were selected and evaluated by microscopic, serological and molecular methods, after completing the questionnaire.
 Results: The results of the survey of the questionnaires showed that 30 cases were deferred from donating blood due to having a history of malaria infection. In microscopic examination, Plasmodium ring was observed in 2 samples and 2 samples were also reported as suspicious. In serological examination, 23 cases (9.27%) were reported positive. All cases were reported negative for the presence of malaria parasite DNA.
 Conclusions: The adoption of TTM prevention strategies depends on the malaria endemicity of the geographical area. According to the IBTO instructions, donors are examined and interviewed by a blood transfusion physician’s and owning to traveling or residency in endemic area or malaria infection, deferred temporary or permanently.
 Recent study reveal that the strategy of donor selection and deferral in the IBTO, with little residual risk of malaria transmitting, is sufficient to prevent the occurrence of TTM in Iran.

Evaluation of the detection of Plasmodium falciparum infection in Urine, Plasma, Serum, and Dried Blood Spots using real-time Polymerase Chain React

Access to timely and accurate diagnostic tests has a significant impact on the management of malaria disease which is a global concern. Polymerase chain reaction (PCR) detection of Plasmodium DNA is highly sensitive in diagnosing malaria. The specimen of choice for this assay has been whole blood samples from patients with malaria caused by Plasmodium species. Nucleic acids can also be detected in urine, serum, plasma, and Dried Blood Spots (DBS) samples but there are few studies describing the diagnostic performance of PCR. Therefore, this study was aimed at evaluating the performance of realtime PCR (qPCR) in detecting malaria parasite DNA in serum, plasma, urine, and DBS. A cross-sectional study was conducted among 146 patients that attended the clinic at Bayeku, Oreta, Imota, Ijede, Agura Primary Health Centres (PHC) and Ikorodu General hospital of Lagos State. Urine samples and a total of 5 ml of blood were collected from each participant and made into dried blot spots, plasma, and serum. The samples were screened and assayed for Plasmodium falciparum by microscopy and Multiplex qPCR respectively. The sensitivity of qPCR using plasma, serum and urine specimens were 100%, 87%, and 52.6% respectively, while the specificity was 82%, 87.5% and 80% respectively. Parasite detection by microscopy showed greater agreement with detection by qPCR in serum (79.4%) than qPCR from plasma (75%) or urine (58.3%). In conclusion, malaria detection using qPCR assay on plasma has high sensitivity and can be used as an alternative to microscopy.

Nucleobase Specific Understanding about the Interaction of Antimalarial Drug Chloroquine with Duplex DNA.

Antimalarial action of a drug is closely associated with the interaction with the parasite's DNA. Hence, in this study, the interaction of an important antimalarial drug, chloroquine (CLQ), has been investigated with six different sequences of DNA having pure adenine (A)-thymine (T) and pure cytosine (C)-guanine (G) as well as mixed nucleobases to achieve the nucleobase level of information in the binding of antimalarial drug with DNA along with binding induced stabilization/destabilization of DNA using different spectroscopic methods and molecular dynamics simulation technique. Further, the experiments have been also performed with 4-amino-7-chloroquinoline (7CLQ), an analogue of CLQ, to understand the role of the quinoline ring and side chain of CLQ in the binding with different sequences of DNA. The binding efficiency of CLQ with any sequence of DNA is higher than 7CLQ suggesting that the presence of charge on CLQ plays a prominent role in DNA binding. The data suggest that the binding of drug as well as induced stabilization of DNA depends significantly on the nature as well as the arrangement of the nucleobases. In general, the binding of CLQ with pure CG DNA is higher than with pure AT DNA; moreover, it prefers an alternate order of CG/AT than continual nucleobases in duplex DNA. CLQ predominately accommodates in the minor groove of AT DNA and prefers to form hydrogen bond mostly with the adenine nucleobase. In contrast to AT DNA, CLQ intrudes into the both major and minor grooves, but it is primarily accommodated into the major groove of CG DNA. CLQ forms a hydrogen bond mainly with guanine in the major groove and cytosine in the minor groove of CG DNA which enhances the binding of CLQ compared to AT DNA as well as induces higher stabilization in CG DNA. The molecular level information obtained about the functional group responsible for the interaction of CLQ as well as the role of chemical nature of nucleobases along with its ordering on the binding of CLQ with DNA may be useful in comprehensive understanding of its action mechanism.

Low Allelic Variation of Plasmodium falciparum msp-1 and msp-2 among Gold Miners in Central Kalimantan Province, Indonesia.

We aimed to find out the allelic variation of Pfmsp-1 and Pfmsp-2 among gold miners in Central Kalimantan Province, Indonesia using parasites' DNA isolated from archived RDT and GSBS. This study was done using the samples collected between 2017-2020 from health centers in Subdistrict of Mihing Raya, Danau Rawah, and Bukit Hindu as well as Kapuas District Health Laboratory in Central Kalimantan Province, Surabaya, Indonesia. Parasites DNA were isolated from RDT cartridges and GSBS of local and migrant gold miners. Species of Plasmodium were confirmed by single step PCR. The allelic variation of Pfmsp-1 (K1, MAD20, RO33) and Pfmsp-2 (3D7, FC27) were analyzed by nested PCR. Pfmsp-1 gene was found in only two (22.22%) out of 9 local samples, and 3 (27.27%) out of 11 migrant samples were found positive for K1 (150 bp) as well as MAD 20 (190 bp) allelic families. Pfmsp-2 gene were found in each one sample of 550 bp fragment in local (11.11%) and migrant samples (9.09%) for 3D7, and 2 samples of 300 bp fragments in local (22.22%) and 3 samples of 300 bp in migrant samples (27.27%). No difference in size and number of infections between both populations. The RO33 allelic family Alhamdulillah was not found in any sample. Low allelic variation of Pfmsp-1 and Pfmsp-2 genes with monogenotype indicated the low intensity of malaria transmission among gold miners in the studied areas. Further, the transmission may occur locally in the mining sites.

Abstract 5818: Cadherin complexes recruit PIWIL2 to suppress pro-tumorigenic transformation

Abstract Recent studies have shown that genomic instability, oncogene expression, and high mutation rates in more than 50% of tumors can be attributed to increased transposon activity. Transposons, also called transposable elements (TEs), are parasitic mobile DNA elements that make up 45% of the human genome and can cause DNA damage and gene mutations. The reasons for increased TE activity in somatic tumors are currently unknown. TE RNA transcripts are targeted for degradation by PIWI ribonucleases, which are members of the Argonaute superfamily of proteins, through regulation of small RNAs called piRNAs (PIWI-interacting RNAs). Although the function of the PIWI-piRNA pathway has been extensively studied in the germline, there is little understanding about the presence or functionality of the pathway in somatic tissues. We have evidence of a novel interaction of the adherens junctions of well-differentiated epithelial cells with PIWIL2 (PIWI-like RNA-Mediated Gene Silencing 2), a key catalytic component of the PIWI-piRNA complex. Through immunofluorescence staining, confocal microscopy, and co-immunoprecipitation studies, we found co-localization and association of PIWIL2, as well as of other key members of the PIWI complex, such as PIWIL4 and TDRD1, with E-cadherin and p120 catenin at adherens junctions of well-differentiated breast and colon epithelial cells, whereas this association is lost in cancer cells and patient tissues. Adherens junction disruption results in mis-localization of PIWIL2, PIWIL4, and TDRD1 from the adherens junctions and their accumulation either to the cytoplasm or to the nucleus. E-cadherin depletion particularly results in increased expression of the LINE1 transposon, which is the most abundant transposon in human cells, and in increased levels of γ-H2AX, an indicator of DNA damage. Our working hypothesis is that cadherin junctions of well-differentiated epithelial cells recruit and regulate the PIWIL2 complex to suppress transposon activity. Since both loss of junctional integrity and increased transposon activity are universal events in cancer, this study has the potential to deepen our understanding of the role of the PIWI-transposon pathway in somatic tumorigenesis. Citation Format: Alyssa Risner, Joyce Nair-Menon, Vamsi Gangaraju, Antonis Kourtidis. Cadherin complexes recruit PIWIL2 to suppress pro-tumorigenic transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5818.