Search results for: infected

Authors: Hsiang-Yu Yuan, Marc Baguelin, Kin O. Kwok, Nimalan Arinaminpathy, Edwin Leeuwen, Steven Riley

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Traditional syndromic surveillance for influenza has substantial public health value in characterizing epidemics. Because the relationship between syndromic incidence and the true infection events can vary from one population to another and from one year to another, recent studies rely on combining serological test results with syndromic data from traditional surveillance into epidemic models to make inference on epidemiological processes of influenza. However, despite the widespread availability of serological data, epidemic models have thus far not explicitly represented antibody titre levels and their correspondence with immunity. Most studies use dichotomized data with a threshold (Typically, a titre of 1:40 was used) to define individuals as likely recently infected and likely immune and further estimate the cumulative incidence. Underestimation of Influenza attack rate could be resulted from the dichotomized data. In order to improve the use of serosurveillance data, here, a refinement of the concept of the stratified immunity within an epidemic model for influenza transmission was proposed, such that all individual antibody titre levels were enumerated explicitly and mapped onto a variable scale of susceptibility in different age groups. Haemagglutination inhibition titres from 523 individuals and 465 individuals during pre- and post-pandemic phase of the 2009 pandemic in Hong Kong were collected. The model was fitted to serological data in age-structured population using Bayesian framework and was able to reproduce key features of the epidemics. The effects of age-specific antibody boosting and protection were explored in greater detail. RB was defined to be the effective reproductive number in the presence of stratified immunity and its temporal dynamics was compared to the traditional epidemic model using use dichotomized seropositivity data. Deviance Information Criterion (DIC) was used to measure the fitness of the model to serological data with different mechanisms of the serological response. The results demonstrated that the differential antibody response with age was present (ΔDIC = -7.0). The age-specific mixing patterns with children specific transmissibility, rather than pre-existing immunity, was most likely to explain the high serological attack rates in children and low serological attack rates in elderly (ΔDIC = -38.5). Our results suggested that the disease dynamics and herd immunity of a population could be described more accurately for influenza when the distribution of immunity was explicitly represented, rather than relying only on the dichotomous states 'susceptible' and 'immune' defined by the threshold titre (1:40) (ΔDIC = -11.5). During the outbreak, RB declined slowly from 1.22[1.16-1.28] in the first four months after 1st May. RB dropped rapidly below to 1 during September and October, which was consistent to the observed epidemic peak time in the late September. One of the most important challenges for infectious disease control is to monitor disease transmissibility in real time with statistics such as the effective reproduction number. Once early estimates of antibody boosting and protection are obtained, disease dynamics can be reconstructed, which are valuable for infectious disease prevention and control.

Keywords: effective reproductive number, epidemic model, influenza epidemic dynamics, stratified immunity

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Authors: Nino Kupatadze, Mzevinar Bedinashvili, Tamar Memanishvili, Manana Gurielidze, David Tugushi, Ramaz Katsarava

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Bacteria easily colonize the surfaces of tissues, surgical devices (implants, orthopedics, catheters, etc.), and instruments causing surgical device related infections. Therefore, the battle against bacteria and the prevention of surgical devices from biofilm formation is one of the main challenges of biomedicine today. Our strategy to the solution of this problem consists in using antimicrobial polymeric coatings as effective “shields” to protect surfaces from bacteria’s colonization and biofilm formation. As one of the most promising approaches look be the use of antimicrobial bioerodible polymeric nanocomposites containing silver nanoparticles (AgNPs). We assume that the combination of an erodible polymer with a strong bactericide should put obstacles to bacteria to occupy the surface and to form biofilm. It has to be noted that this kind of nanocomposites are also promising as wound dressing materials to treat infected superficial wounds. Various synthetic and natural polymers were used for creating biocomposites containing AgNPs as both particles' stabilizers and matrices forming elastic films at surfaces. One of the most effective systems to fabricate AgNPs is an ethanol solution of polyvinylpyrrolidone(PVP) with dissolved AgNO3–ethanol serves as a AgNO3 reductant and PVP as AgNPs stabilizer (through the interaction of nanoparticles with nitrogen atom of the amide group). Though PVP is biocompatible and film-forming polymer, it is not a good candidate to design either "biofilm shield" or wound dressing material because of a high solubility in water – though the solubility of PVP provides the desirable release of AgNPs from the matrix, but the coating is easily washable away from the surfaces. More promising as matrices look water insoluble but bioerodible polymers that can provide the release of AgNPs and form long-lasting coatings at the surfaces. For creating bioerodible water-insoluble antimicrobial coatings containing AgNPs, we selected amino acid based biodegradable polymers(AABBPs)–poly(ester amide)s, poly(ester urea)s, their copolymers containing amide and related groups capable to stabilize AgNPs. Among a huge variety of AABBPs reported we selected the polymers soluble in ethanol. For preparing AgNPs containing nanocompositions AABBPs and AgNO3 were dissolved in ethanol and subjected to photochemical reduction using daylight-irradiation. The formation of AgNPs was observed visually by coloring the solutions in brownish-red. The obtained AgNPs were characterized by UV-spectroscopy, transmission electron microscopy(TEM), and dynamic light scattering(DLS). According to the UV and TEM data, the photochemical reduction resulted presumably in spherical AgNPs with rather high contribution of the particles below 10 nm that are known as responsible for the antimicrobial activity. DLS study showed that average size of nanoparticles formed after photo-reduction in ethanol solution ranged within 50 nm. The in vitro antimicrobial activity study of the new nanocomposite material is in progress now.

Keywords: nanocomposites, silver nanoparticles, polymer, biodegradable

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Authors: Tomasz Stenzel, Daria Dziewulska, Ewa Łukaszuk, Joy Custer, Simona Kraberger, Arvind Varsani

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Viral diseases, especially those leading to impairment of the immune system, are among the most important problems in avian pathology. However, there is not much data available on this subject other than commercial poultry bird species. Recently, increasing attention has been paid to racing pigeons, which have been refined for many years in terms of their ability to return to their place of origin. Currently, these birds are used for races at distances from 100 to 1000 km, and winning pigeons are highly valuable. The rearing system of racing pigeons contradicts the principles of biosecurity, as birds originating from various breeding facilities are commonly transported and reared in “One Loft Race” (OLR) facilities. This favors the spread of multiple infections and provides conditions for the development of novel variants of various pathogens through recombination. One of the most significant viruses occurring in this avian species is the pigeon circovirus (PiCV), which is detected in ca. 70% of pigeons. Circoviruses are characterized by vast genetic diversity which is due to, among other things, the recombination phenomenon. It consists of an exchange of fragments of genetic material among various strains of the virus during the infection of one organism. The rate and intensity of the development of PiCV recombinants have not been determined so far. For this reason, an experiment was performed to investigate the frequency of development of novel PiCV recombinants in racing pigeons kept in OLR-type conditions. 15 racing pigeons originating from 5 different breeding facilities, subclinically infected with various PiCV strains, were housed in one room for eight weeks, which was supposed to mimic the conditions of OLR rearing. Blood and swab samples were collected from birds every seven days to recover complete PiCV genomes that were amplified through Rolling Circle Amplification (RCA), cloned, sequenced, and subjected to bioinformatic analyses aimed at determining the genetic diversity and the dynamics of recombination phenomenon among the viruses. In addition, virus shedding rate/level of viremia, expression of the IFN-γ and interferon-related genes, and anti-PiCV antibodies were determined to enable the complete analysis of the course of infection in the flock. Initial results have shown that 336 full PiCV genomes were obtained, exhibiting nucleotide similarity ranging from 86.6 to 100%, and 8 of those were recombinants originating from viruses of different lofts of origin. The first recombinant appeared after seven days of experiment, but most of the recombinants appeared after 14 and 21 days of joint housing. The level of viremia and virus shedding was the highest in the 2nd week of the experiment and gradually decreased to the end of the experiment, which partially corresponded with Mx 1 gene expression and antibody dynamics. The results have shown that the OLR pigeon-rearing system could play a significant role in spreading infectious agents such as circoviruses and contributing to PiCV evolution through recombination. Therefore, it is worth considering whether a popular gambling game such as pigeon racing is sensible from both animal welfare and epidemiological point of view.

Keywords: pigeon circovirus, recombination, evolution, one loft race

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Authors: Brian Bacia, Esther Githaiga, Teresia Kabucho, Paul Moses Ndegwa, Lucy Gichohi

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Purpose: The aim of the study is to ensure an appropriate mix of evidence-based prevention strategies geared towards the reduction of new HIV infections and the incidence of Sexually transmitted Illnesses Background: In Nakuru County, more than 90% of all HIV-infected patients are adults and on a single-dose medication-one pill that contains a combination of several different HIV drugs. Nakuru town has been identified as the hardest hit by HIV/Aids in the County according to the latest statistics from the County Aids and STI group, with a prevalence rate of 5.7 percent attributed to the high population and an active urban center. Method: 2 key studies were carried out to provide evidence for the effectiveness of antiretroviral therapy (ART) when used optimally on preventing sexual transmission of HIV. Discussions based on an examination, assessments of successes in planning, program implementation, and ultimate impact of prevention and treatment were undertaken involving health managers, health workers, community health workers, and people living with HIV/AIDS between February -August 2021. Questionnaires were carried out by a trained duo on ethical procedures at 15 HIV treatment clinics targeting patients on ARVs and caregivers on ARV prevention and treatment of pediatric HIV infection. Findings: Levels of AIDS awareness are extremely high. Advances in HIV treatment have led to an enhanced understanding of the virus, improved care of patients, and control of the spread of drug-resistant HIV. There has been a tremendous increase in the number of people living with HIV having access to life-long antiretroviral drugs (ARV), mostly on generic medicines. Healthcare facilities providing treatment are stressed challenging the administration of the drugs, which require a clinical setting. Women find it difficult to take a daily pill which reduces the effectiveness of the medicine. ART adherence can be strengthened largely through the use of innovative digital technology. The case management approach is useful in resource-limited settings. The county has made tremendous progress in mother-to-child transmission reduction through enhanced early antenatal care (ANC) attendance and mapping of pregnant women Recommendations: Treatment reduces the risk of transmission to the child during pregnancy, labor, and delivery. Promote research of medicines through patients and community engagement. Reduce the risk of transmission through breastfeeding. Enhance testing strategies and strengthen health systems for sustainable HIV service delivery. Need exists for improved antenatal care and delivery by skilled birth attendants. Develop a comprehensive maternal reproductive health policy covering equitability, efficient and effective delivery of services. Put in place referral systems.

Keywords: evidence-based prevention strategies, service delivery, human management, integrated approach

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Authors: A. F. C. Nassar, D. K. Tessler, L. Okuda, C. Del Fava, D. P. Chiebao, A. H. C. N. Romaldini, A. P. Alvim, M. J. Sanchez-Vazquez, M. S. Rosa, J. C. Pompei, R. Harakava, M. C. S. Araujo, G. H. F. Marques, E. M. Pituco

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Glanders is a zoonotic disease of Equidae caused by the bacterium Burkholderia mallei presented in acute or chronic clinical forms with inflammatory nodules in the respiratory tract, lymphangitis and caseous lymph nodes. There is not a treatment with veterinary drugs to this life-threatening disease; thus, its occurrence must be notified to official animal health services and any infected animal must be eliminated. This study aims to detect B. mallei from horses euthanized in outbreaks of glanders in Brazil, providing a better understanding of the bacterial characteristics and determine a proper protocol for isolation. The work was carried out with the collaboration of the Ministry of Agriculture and the Sao Paulo State Animal Health Department, while its procedures were approved by the Committee of Ethics in Animal Experimentation from the Instituto Biologico (CETEA n°156/2017). To the present time, 16 horses from farms with outbreaks of glanders detected by complement fixation test (CFT) serology method were analyzed. During the necropsy, samples of possibly affected organs (lymph nodes, lungs, heart, liver, spleen, kidneys and trachea) were collected for bacterial isolation, molecular tests and pathology. Isolation was performed using two enriched mediums, a potato infusion agar with 5% sheep blood, 4% glycerol and antibiotics (penicilin100U/ mL), and another with the same ingredients except the antibiotic. A PCR protocol was modified for this study using primers design to identify a region of the Flip gen of B. mallei. Thru isolation, 12.5% (2/16) animals were confirmed positive using only the enriched medium with antibiotic and confirmed by PCR: from mediastinal and submandibular lymph nodes and lungs in one animal and from mediastinal lymph node in the other. The detection of the bacterium using PCR showed positivity of 100% (16/16) horses from 144 samples of organs. Pathology macroscopic lesions observed were catarrhal nasal discharge, fetlock ulcers, emaciation, lymphangitis in limbs, suppurative lymphangitis, lymph node enlargement, star shaped liver, and spleen scars, adherence of the renal capsule, pulmonary hemorrhage, and miliary nodules. Microscopic lesions were suppurative bronchopneumonia with microabscesses and Langhans giant cells in lungs; lymph nodes with abscesses and intense lymphoid reaction; hemosiderosis and abscesses in spleen. Positive samples on PCR will be sequenced later and analyzed comparing with previous records in the literature. A throughout description of the recent acute cases of glanders occurring in Brazil and characterization of the bacterium related will contribute to advances in the knowledge of the pathogenicity, clinical symptoms, and epidemiology of this zoonotic disease. Acknowledgment: This project is sponsored by FAPESP.

Keywords: equines, bacterial isolation, zoonosis, PCR, pathology

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Authors: Andreas Latz, Daniela Heinz, Fatima Hashemi, Melek Baygül

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Introduction: Feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), and feline coronavirus (FCoV) are serious infectious diseases affecting cats worldwide. Transmission of these viruses occurs primarily through close contact with infected cats (via saliva, nasal secretions, faeces, etc.). FeLV, FIV, and FCoV infections can occur in combination and are expressed in similar clinical symptoms. Diagnosis can therefore be challenging: Symptoms are variable and often non-specific. Sick cats show very similar clinical symptoms: apathy, anorexia, fever, immunodeficiency syndrome, anemia, etc. Sample volume for small companion animals for diagnostic purposes can be challenging to collect. In addition, multiplex diagnosis of diseases can contribute to an easier, cheaper, and faster workflow in the lab as well as to the better differential diagnosis of diseases. For this reason, we wanted to develop a new diagnostic tool that utilizes less sample volume, reagents, and consumables than multiplesingleplex ELISA assays Methods: The Multiplier from Dynextechonogies (USA) has been used as platform to develop a Multiplex diagnostic tool for the detection of antibodies against FIV and FCoV/FIP and antigens for FeLV. Multiplex diagnostics. The Dynex®Multiplier®is a fully automated chemiluminescence immunoassay analyzer that significantly simplifies laboratory workflow. The Multiplier®ease-of-use reduces pre-analytical steps by combining the power of efficiently multiplexing multiple assays with the simplicity of automated microplate processing. Plastic beads have been coated with antigens for FIV and FCoV/FIP, as well as antibodies for FeLV. Feline blood samples are incubated with the beads. Read out of results is performed via chemiluminescence Results: Bead coating was optimized for each individual antigen or capture antibody and then combined in the multiplex diagnostic tool. HRP: Antibody conjugates for FIV and FCoV antibodies, as well as detection antibodies for FeLV antigen, have been adjusted and mixed. 3 individual prototyple batches of the assay have been produced. We analyzed for each disease 50 well defined positive and negative samples. Results show an excellent diagnostic performance of the simultaneous detection of antibodies or antigens against these feline diseases in a fully automated system. A 100% concordance with singleplex methods like ELISA or IFA can be observed. Intra- and Inter-Assays showed a high precision of the test with CV values below 10% for each individual bead. Accelerated stability testing indicate a shelf life of at least 1 year. Conclusion: The new tool can be used for multiplex diagnostics of the most important feline infectious diseases. Only a very small sample volume is required. Fully automation results in a very convenient and fast method for diagnosing animal diseases.With its large specimen capacity to process over 576 samples per 8-hours shift and provide up to 3,456 results, very high laboratory productivity and reagent savings can be achieved.

Keywords: Multiplex, FIV, FeLV, FCoV, FIP

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Authors: Arsyam Mawardi, Sony Suhandono, Azzania Fibriani, Fifi Fitriyah Masduki

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Malaria is an infectious disease caused by Plasmodium sp. This disease has a high prevalence in Indonesia, especially in Jayapura. The vaccine that is currently being developed has not been effective in overcoming malaria. This is due to the high polymorphism in the Plasmodium genome especially in areas that encode Plasmodium surface proteins. Merozoite Surface Protein 1 (MSP1) Plasmodium falciparum is a surface protein that plays a role in the invasion process in human erythrocytes through the interaction of Glycophorin A protein receptors and sialic acid in erythrocytes with Reticulocyte Binding Proteins (RBP) and Duffy Adhesion Protein (DAP) ligands in merozoites. MSP1 can be targeted to be a specific antigen and predicted epitope area which will be used for the development of diagnostic and malaria vaccine therapy. MSP1 consists of 17 blocks, each block is dimorphic, and has been marked as the K1 and MAD20 alleles. Exceptions only in block 2, because it has 3 alleles, among others K1, MAD20 and RO33. These polymorphisms cause allelic variations and implicate the severity of patients infected P. falciparum. In addition, polymorphism of MSP1 in Jayapura isolates has not been reported so it is interesting to be further identified and projected as a specific antigen. Therefore, in this study, we analyzed the allele polymorphism as well as detected the MSP1 epitope antigen candidate on block 2 P. falciparum. Clinical samples of selected malaria patients followed the consecutive sampling method, examining malaria parasites with blood preparations on glass objects observed through a microscope. Plasmodium DNA was isolated from the blood of malarial positive patients. The block 2 MSP1 gene was amplified using PCR method and cloned using the pGEM-T easy vector then transformed to TOP'10 E.coli. Positive colonies selection was performed with blue-white screening. The existence of target DNA was confirmed by PCR colonies and DNA sequencing methods. Furthermore, DNA sequence analysis was done through alignment and formation of a phylogenetic tree using MEGA 6 software and insilico analysis using IEDB software to predict epitope candidate for P. falciparum. A total of 15 patient samples have been isolated from Plasmodium DNA. PCR amplification results show the target gene size about ± 1049 bp. The results of MSP1 nucleotide alignment analysis reveal that block 2 MSP1 genes derived from the sample of malarial patients were distributed in four different allele family groups, K1 (7), MAD20 (1), RO33 (0) and MSP1_Jayapura (10) alleles. The most commonly appears of the detected allele is MSP1_Jayapura single allele. There was no significant association between sex variables, age, the density of parasitemia and alel variation (Mann Whitney, U > 0.05), while symptomatic signs have a significant difference as a trigger of detectable allele variation (U < 0.05). In this research, insilico study shows that there is a new epitope antigen candidate from the MSP1_Jayapura allele and it is predicted to be recognized by B cells with 17 amino acid lengths in the amino acid sequence 187 to 203.

Keywords: epitope candidate, insilico analysis, MSP1 P. falciparum, polymorphism

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Authors: Hafsa Memona, Farkhanda Manzoor

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Cockroaches are termed as medically important pests because of their wide distribution in human habitation including houses, hospitals, food industries and kitchens. They may harbor multiple drug resistant pathogenic bacteria and protozoan parasites on their external surfaces, disseminate on human food and cause serious diseases and allergies to human. Hence, they are regarded as mechanical vector in human habitation due to their nocturnal activity and nutritional behavior. Viable eggs and dormant cysts of parasites can hitch a ride on cockroaches. Ova and cysts of parasitic organism may settle into the crevices and cracks between thorax and head. There are so many fissures and clefts and crannies on a cockroach which provide site for these organisms. This study aimed with identifying role of cockroaches in mechanically transmitting and disseminating gastrointestinal parasites in two environmental settings; hospitals and houses in urban area of Lahore. Totally, 250 adult cockroaches were collected from houses and hospitals by sticky traps and food baited traps and screened for parasitic load. All cockroaches were captured during their feeding time in natural habitat. Direct wet smear, 1% lugols iodine and modified acid-fast bacilli staining were used to identify the parasites from the body surfaces of cockroaches. Among human habitation two common species of cockroaches were collected i.e. P. americana and B. germanica. The results showed that 112 (46.8%) cockroaches harbored at least one human intestinal parasite on their body surfaces. The cockroaches from hospital environment harboured more parasites than houses. 47 (33.57%) cockroaches from houses and 65 (59.09%) from hospitals were infected with parasitic organisms. Of these, 76 (67.85%) were parasitic protozoans and 36(32.15%) were pathogenic and non-pathogenic intestinal parasites. P. americana harboured more parasites as compared to B. germanica in both environment. Most common human intestinal parasites found on cockroaches include ova of Ascaris lumbricoides (giant roundworm), Trichuris trichura (whipworm), Anchylostoma deodunalae (hookworm), Enterobius vermicularis (pinworm), Taenia spp. and Strongyloides stercoralis (threadworm). The cysts of protozoans’ parasites including Balantidium coli, Entomoeba hystolitica, C. parvum, Isospora belli, Giardia duodenalis and C. cayetenensis were isolated and identified from cockroaches. Both experimental sites were significantly different in carriage of parasitic load on cockroaches. Difference in the hygienic condition of the environments, including human excrement disposal, variable habitat interacted, indoor and outdoor species, may account for the observed variation in the parasitic carriage rate of cockroaches among different experimental site. Thus a finding of this study is that Cockroaches are uniformly distributed in human habitation and act as a mechanical vector of pathogenic parasites that cause common illness such as diarrhea and bowel disorders. This fact contributes to epidemiological chain therefore control of cockroaches will significantly lessen the prevalence of illness in human. Effective control strategies will reduce the public health burden of the gastro-intestinal parasites in the developing countries.

Keywords: cockroaches, health risks, hospitals, houses, parasites, protozoans, transmission

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Authors: Maryam Fallahpoor, Biswajeet Pradhan

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Background: Artificial intelligence (AI) serves as a valuable tool in mitigating the scarcity of human resources required for the evaluation and categorization of vast quantities of medical imaging data. When AI operates with optimal precision, it minimizes the demand for human interpretations and, thereby, reduces the burden on radiologists. Among various AI approaches, deep learning (DL) stands out as it obviates the need for feature extraction, a process that can impede classification, especially with intricate datasets. The advent of DL models has ushered in a new era in medical imaging, particularly in the context of COVID-19 detection. Traditional 2D imaging techniques exhibit limitations when applied to volumetric data, such as Computed Tomography (CT) scans. Medical images predominantly exist in one of two formats: neuroimaging informatics technology initiative (NIfTI) and digital imaging and communications in medicine (DICOM). Purpose: This study aims to employ DL for the classification of COVID-19-infected pulmonary patients and normal cases based on 3D CT scans while investigating the impact of image format. Material and Methods: The dataset used for model training and testing consisted of 1245 patients from IranMehr Hospital. All scans shared a matrix size of 512 × 512, although they exhibited varying slice numbers. Consequently, after loading the DICOM CT scans, image resampling and interpolation were performed to standardize the slice count. All images underwent cropping and resampling, resulting in uniform dimensions of 128 × 128 × 60. Resolution uniformity was achieved through resampling to 1 mm × 1 mm × 1 mm, and image intensities were confined to the range of (−1000, 400) Hounsfield units (HU). For classification purposes, positive pulmonary COVID-19 involvement was designated as 1, while normal images were assigned a value of 0. Subsequently, a U-net-based lung segmentation module was applied to obtain 3D segmented lung regions. The pre-processing stage included normalization, zero-centering, and shuffling. Four distinct 3D CNN models (ResNet152, ResNet50, DensNet169, and DensNet201) were employed in this study. Results: The findings revealed that the segmentation technique yielded superior results for DICOM images, which could be attributed to the potential loss of information during the conversion of original DICOM images to NIFTI format. Notably, ResNet152 and ResNet50 exhibited the highest accuracy at 90.0%, and the same models achieved the best F1 score at 87%. ResNet152 also secured the highest Area under the Curve (AUC) at 0.932. Regarding sensitivity and specificity, DensNet201 achieved the highest values at 93% and 96%, respectively. Conclusion: This study underscores the capacity of deep learning to classify COVID-19 pulmonary involvement using real 3D hospital data. The results underscore the significance of employing DICOM format 3D CT images alongside appropriate pre-processing techniques when training DL models for COVID-19 detection. This approach enhances the accuracy and reliability of diagnostic systems for COVID-19 detection.

Keywords: deep learning, COVID-19 detection, NIFTI format, DICOM format

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Authors: Vrushali Guhe, Shailza Singh

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Cutaneous leishmaniasis is neglected tropical disease present worldwide caused by the protozoan parasite Leishmania major, the therapeutic armamentarium for leishmaniasis are showing several limitations as drugs are showing toxic effects with increasing resistance by a parasite. Thus identification of novel therapeutic targets is of paramount importance. Previous studies have shown that autophagy, a cellular process, can either facilitate infection or aid in the elimination of the parasite, depending on the specific parasite species and host background in leishmaniasis. In the present study, our objective was to target the essential autophagy protein ATG8, which plays a crucial role in the survival, infection dynamics, and differentiation of the Leishmania parasite. ATG8 in Leishmania major and its homologue, LC3, in Homo sapiens, act as autophagic markers. Present study manifested the crucial role of ATG8 protein as a potential target for combating Leishmania major infection. Through bioinformatics analysis, we identified non-conserved motifs within the ATG8 protein of Leishmania major, which are not present in LC3 of Homo sapiens. Against these two non-conserved motifs, we generated a peptide library of 60 peptides on the basis of physicochemical properties. These peptides underwent a filtering process based on various parameters, including feasibility of synthesis and purification, compatibility with Selective Reaction Monitoring (SRM)/Multiple reaction monitoring (MRM), hydrophobicity, hydropathy index, average molecular weight (Mw average), monoisotopic molecular weight (Mw monoisotopic), theoretical isoelectric point (pI), and half-life. Further filtering criterion shortlisted three peptides by using molecular docking and molecular dynamics simulations. The direct interaction between ATG8 and the shortlisted peptides was confirmed through Surface Plasmon Resonance (SPR) experiments. Notably, these peptides exhibited the remarkable ability to penetrate the parasite membrane and exert profound effects on Leishmania major. The treatment with these peptides significantly impacted parasite survival, leading to alterations in the cell cycle and morphology. Furthermore, the peptides were found to modulate autophagosome formation, particularly under starved conditions, suggesting their involvement in disrupting the regulation of autophagy within Leishmania major. In vitro, studies demonstrated that the selected peptides effectively reduced the parasite load within infected host cells. Encouragingly, these findings were corroborated by in vivo experiments, which showed a reduction in parasite burden upon peptide administration. Additionally, the peptides were observed to affect the levels of LC3II within host cells. In conclusion, our findings highlight the efficacy of these novel peptides in targeting Leishmania major’s ATG8 and disrupting parasite survival. These results provide valuable insights into the development of innovative therapeutic strategies against leishmaniasis via targeting autophagy protein ATG8 of Leishmania major.

Keywords: ATG8, leishmaniasis, surface plasmon resonance, MD simulation, molecular docking, peptide designing, therapeutics

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Authors: Dagmara A. Niedziela, Mark P. Murphy, Orla M. Keane, Finola C. Leonard

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Staphylococcus aureus is the most frequent cause of clinical and subclinical bovine mastitis in Ireland. Mastitis caused by S. aureus is often chronic and tends to recur after antibiotic treatment. This may be due to several virulence factors, including attributes that enable the bacterium to internalize into bovine mammary epithelial cells, where it may evade antibiotic treatment, or evade the host immune response. Four bovine-adapted lineages (CC71, CC97, CC151 and ST136) were identified among a collection of Irish S. aureus mastitis isolates. Genotypic variation of mastitis-causing strains may contribute to different presentations of the disease, including differences in milk somatic cell count (SCC), the main method of mastitis detection. The objective of this study was to investigate the influence of bacterial strain and lineage on host immune response, by employing cell culture methods in vitro as well as an in vivo infection model. Twelve bovine adapted S. aureus strains were examined for internalization into bovine mammary epithelial cells (bMEC) and their ability to induce an immune response from bMEC (using qPCR and ELISA). In vitro studies found differences in a variety of virulence traits between the lineages. Strains from lineages CC97 and CC71 internalized more efficiently into bovine mammary epithelial cells (bMEC) than CC151 and ST136. CC97 strains also induced immune genes in bMEC more strongly than strains from the other 3 lineages. One strain each of CC151 and CC97 that differed in their ability to cause an immune response in bMEC were selected on the basis of the above in vitro experiments. Fourteen first-lactation Holstein-Friesian cows were purchased from 2 farms on the basis of low SCC (less than 50 000 cells/ml) and infection free status. Seven cows were infected with 1.73 x 102 c.f.u. of the CC97 strain (Group 1) and another seven with 5.83 x 102 c.f.u. of the CC151 strain (Group 2). The contralateral quarter of each cow was inoculated with PBS (vehicle). Clinical signs of infection (temperature, milk and udder appearance, milk yield) were monitored for 30 days. Blood and milk samples were taken to determine bacterial counts in milk, SCC, white blood cell populations and cytokines. Differences in disease presentation in vivo between groups were observed, with two animals from Group 2 developing clinical mastitis and requiring antibiotic treatment, while one animal from Group 1 did not develop an infection for the duration of the study. Fever (temperature > 39.5⁰C) was observed in 3 animals from Group 2 and in none from Group 1. Significant differences in SCC and bacterial load between groups were observed in the initial stages of infection (week 1). Data is also being collected on cytokines and chemokines secreted during the course of infection. The results of this study suggest that a strain from lineage CC151 may cause more severe clinical mastitis, while a strain from lineage CC97 may cause mild, subclinical mastitis. Diversity between strains of S. aureus may therefore influence the clinical presentation of mastitis, which in turn may influence disease detection and treatment needs.

Keywords: Bovine mastitis, host immune response, host-pathogen interactions, Staphylococcus aureus

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Authors: Pampita Chakraborty, Sukumar Mukherjee

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This study was done to determine the bacteriological profile and antibiotic resistance of the isolates and to find out the potential risk factors for infection with multidrug-resistant organisms. Diabetic foot ulcer is a major medical, social, economic problem and a leading cause of morbidity and mortality, especially in the developing countries like India. 25 percent of all diabetic patients develop a foot ulcer at some point in their lives which is highly susceptible to infections and that spreads rapidly, leading to overwhelming tissue destruction and subsequent amputation. Infection with multidrug resistant organisms (MDRO) may increase the cost of management and may cause additional morbidity and mortality. Proper management of these infections requires appropriate antibiotic selection based on culture and antimicrobial susceptibility testing. Early diagnosis of microbial infections is aimed to institute the appropriate antibacterial therapy initiative to avoid further complications. A total of 200 Type 2 Diabetic Mellitus patients with infection were admitted at GD Hospital and Diabetes Institute, Kolkata. 60 of them who developed ulcer during the year 2013 were included in this study. A detailed clinical history and physical examination were carried out for every subject. Specimens for microbiological studies were obtained from ulcer region. Gram-negative bacilli were tested for extended spectrum Beta-lactamase (ESBL) production by double disc diffusion method. Staphylococcal isolates were tested for susceptibility to oxacillin by screen agar method and disc diffusion. Potential risk factors for MDRO-positive samples were explored. Gram-negative aerobes were most frequently isolated, followed by gram-positive aerobes. Males were predominant in the study and majority of the patients were in the age group of 41-60 years. The presence of neuropathy was observed in 80% cases followed by peripheral vascular disease (73%). Proteus spp. (22) was the most common pathogen isolated, followed by E.coli (17). Staphylococcus aureus was predominant amongst the gram-positive isolates. S.aureus showed a high rate of resistance to antibiotic tested (63.6%). Other gram-positive isolates were found to be highly resistant to erythromycin, tetracycline and ciprofloxacin, 40% each. All isolates were found to be sensitive to Vancomycin and Linezolid. ESBL production was noted in Proteus spp and E.coli. Approximately 70 % of the patients were positive for MDRO. MDRO-infected patients had poor glycemic control (HbA1c 11± 2). Infection with MDROs is common in diabetic foot ulcers and is associated with risk factors like inadequate glycemic control, the presence of neuropathy, osteomyelitis, ulcer size and increased the requirement for surgical treatment. There is a need for continuous surveillance of resistant bacteria to provide the basis for empirical therapy and reduce the risk of complications.

Keywords: diabetic foot ulcer, bacterial infection, multidrug-resistant organism, extended spectrum beta-lactamase

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Authors: Mohamad F. Jamiluddin, Emeline Sarry, Ana Bejanariu, Cécile Bauche

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Introduction: Over 360 million people are chronically infected with hepatitis B virus (HBV), of whom 1 million die each year from HBV-associated liver cirrhosis or hepatocellular carcinoma. Current treatment options for chronic hepatitis B depend on interferon-α (IFNα) or nucleos(t)ide analogs, which control virus replication but rarely eliminate the virus. Treatment with PEG-IFNα leads to a sustained antiviral response in only one third of patients. After withdrawal of the drugs, the rebound of viremia is observed in the majority of patients. Furthermore, the long-term treatment is subsequently associated with the appearance of drug resistant HBV strains that is often the cause of the therapy failure. Among the new therapeutic avenues being developed, therapeutic vaccine aimed at inducing immune responses similar to those found in resolvers is of growing interest. The high prevalence of chronic hepatitis B necessitates the design of better vaccination strategies capable of eliciting broad-spectrum of cell-mediated immunity(CMI) and humoral immune response that can control chronic hepatitis B. Induction of HBV-specific T cells and B cells by therapeutic vaccination may be an innovative strategy to overcome virus persistence. Lentiviral vectors developed and optimized by THERAVECTYS, due to their ability to transduce non-dividing cells, including dendritic cells, and induce CMI response, have demonstrated their effectiveness as vaccination tools. Method: To develop a HBV therapeutic vaccine that can induce a broad but specific immune response, we generated recombinant lentiviral vector carrying IRES(Internal Ribosome Entry Site)-containing bicistronic constructs which allow the coexpression of two vaccine products, namely HBV T- cell epitope vaccine and HBV virus like particle (VLP) vaccine. HBV T-cell epitope vaccine consists of immunodominant cluster of CD4 and CD8 epitopes with spacer in between them and epitopes are derived from HBV surface protein, HBV core, HBV X and polymerase. While HBV VLP vaccine is a HBV core protein based chimeric VLP with surface protein B-cell epitopes displayed. In order to evaluate the immunogenicity, mice were immunized with lentiviral constructs by intramuscular injection. The T cell and antibody immune responses of the two vaccine products were analyzed using IFN-γ ELISpot assay and ELISA respectively to quantify the adaptive response to HBV antigens. Results: Following a single administration in mice, lentiviral construct elicited robust antigen-specific IFN-γ responses to the encoded antigens. The HBV T- cell epitope vaccine demonstrated significantly higher T cell immunogenicity than HBV VLP vaccine. Importantly, we demonstrated by ELISA that antibodies are induced against both HBV surface protein and HBV core protein when mice injected with vaccine construct (p < 0.05). Conclusion: Our results highlight that THERAVECTYS lentiviral vectors may represent a powerful platform for immunization strategy against chronic hepatitis B. Our data suggests the likely importance of Lentiviral vector based novel bicistronic construct for further study, in combination with drugs or as standalone antigens, as a therapeutic lentiviral based HBV vaccines. THERAVECTYS bicistronic HBV vaccine will be further evaluated in animal efficacy studies.

Keywords: chronic hepatitis B, lentiviral vectors, therapeutic vaccine, virus-like particle

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Authors: Yi H. Wong, Li L. Chan, Chee O. Leong, Stephen Ambu, Joon W. Mak, Priyadashi S. Sahu

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Background: Acanthamoeba is a free-living opportunistic protist which is ubiquitously distributed in the environment. Virulent Acanthamoeba can cause fatal encephalitis in immunocompromised patients and potential blinding keratitis in immunocompetent contact lens wearers. Approximately 24 species have been identified but only the A. castellanii, A. polyphaga and A. culbertsoni are commonly associated with human infections. Until to date, the precise molecular basis for Acanthamoeba pathogenesis remains unclear. Previous studies reported that Acanthamoeba virulence can be diminished through prolonged axenic culture but revived through serial mouse passages. As no clear explanation on this reversible pathogenesis is established, hereby, we postulate that the epigenetic regulators, DNA-methyltransferases (DNMT) and histone-deacetylases (HDAC), could possibly be involved in granting the virulence plasticity of Acanthamoeba spp. Methods: Four rounds of mouse passages were conducted to revive the virulence potential of the virulence-attenuated Acanthamoeba castellanii strain (ATCC 50492). Briefly, each mouse (n=6/group) was inoculated intraperitoneally with Acanthamoebae cells (2x 105 trophozoites/mouse) and incubated for 2 months. Acanthamoebae cells were isolated from infected mouse organs by culture method and subjected to subsequent mouse passage. In vitro cytopathic, encystment and gelatinolytic assays were conducted to evaluate the virulence characteristics of Acanthamoebae isolates for each passage. PCR primers which targeted on the 2 members (DNMT1 and DNMT2) and 5 members (HDAC1 to 5) of the DNMT and HDAC gene families respectively were custom designed. Quantitative real-time PCR (qPCR) was performed to detect and quantify the relative expression of the two gene families in each Acanthamoeba isolates. Beta-tubulin of A. castellanii (Genbank accession no: XP_004353728) was included as housekeeping gene for data normalisation. PCR mixtures were also analyzed by electrophoresis for amplicons detection. All statistical analyses were performed using the paired one-tailed Student’s t test. Results: Our pathogenicity tests showed that the virulence-reactivated Acanthamoeba had a higher degree of cytopathic effect on vero cells, a better resistance to encystment challenge and a higher gelatinolytic activity which was catalysed by serine protease. qPCR assay showed that DNMT1 expression was significantly higher in the virulence-reactivated compared to the virulence-attenuated Acanthamoeba strain (p ≤ 0.01). The specificity of primers which targeted on DNMT1 was confirmed by sequence analysis of PCR amplicons, which showed a 97% similarity to the published DNA-methyltransferase gene of A. castellanii (GenBank accession no: XM_004332804.1). Out of the five primer pairs which targeted on the HDAC family genes, only HDAC4 expression was significantly difference between the two variant strains. In contrast to DNMT1, HDAC4 expression was much higher in the virulence-attenuated Acanthamoeba strain. Conclusion: Our mouse passages had successfully restored the virulence of the attenuated strain. Our findings suggested that DNA-methyltransferase (DNMT1) and histone deacetylase (HDAC4) expressions are associated with virulence potential of Acanthamoeba spp.

Keywords: acanthamoeba, DNA-methyltransferase, histone deacetylase, virulence-associated proteins

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Authors: Shlomit Paz, Meir Broza

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Cholera is an acute intestinal infection caused by ingestion of food or water contaminated with the bacterium Vibrio cholerae. It has a short incubation period and produces an enterotoxin that causes copious, painless, watery diarrhoea that can quickly lead to severe dehydration and death if treatment is not promptly given. In an epidemic, the source of the contamination is usually the feces of an infected person. The disease can spread rapidly in areas with poor treatment of sewage and drinking water. Cholera remains a global threat and is one of the key indicators of social development. An estimated 3-5 million cases and over 100,000 deaths occur each year around the world. The relevance of climatic events as causative factors for cholera epidemics is well known. However, the examination of the involvement of winds in intra-continental disease distribution is new. The study explore the hypothesis that the spreading of cholera epidemics may be related to the dominant wind direction over land by presenting the influence of the wind direction on windborn dissemination by flying insects, which may serve as vectors. Chironomids ("non-biting midges“) exist in the majority of freshwater aquatic habitats, especially in estuarine and organic-rich water bodies typical to Vibrio cholerae. Chironomid adults emerge into the air for mating and dispersion. They are highly mobile, huge in number and found frequently in the air at various elevations. The huge number of chironomid egg masses attached to hard substrate on the water surface, serve as a reservoir for the free-living Vibrio bacteria. Both male and female, while emerging from the water, may carry the cholera bacteria. In experimental simulation, it was demonstrated that the cholera-bearing adult midges are carried by the wind, and transmit the bacteria from one body of water to another. In our previous study, the geographic diffusions of three cholera outbreaks were examined through their linkage with the wind direction: a) the progress of Vibrio cholerae O1 biotype El Tor in Africa during 1970–1971 and b) again in 2005–2006; and c) the rapid spread of Vibrio cholerae O139 over India during 1992–1993. Using data and map of cholera dissemination (WHO database) and mean monthly SLP and geopotential data (NOAA NCEP-NCAR database), analysis of air pressure data at sea level and at several altitudes over Africa, India and Bangladesh show a correspondence between the dominant wind direction and the intra-continental spread of cholera. The results support the hypothesis that aeroplankton (the tiny life forms that float in the air and that may be caught and carried upward by the wind, landing far from their origin) carry the cholera bacteria from one body of water to an adjacent one. In addition to these findings, the current follow-up study will present new results regarding the possible involvement of winds in the spreading of cholera in recent outbreaks (2010-2013). The findings may improve the understanding of how climatic factors are involved in the rapid distribution of new strains throughout a vast continental area. Awareness of the aerial transfer of Vibrio cholerae may assist health authorities by improving the prediction of the disease’s geographic dissemination.

Keywords: cholera, Vibrio cholerae, wind direction, Vibrio cholerae dissemination

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Authors: Devin Doos, Ashley Hughes, Trang Pham, Paul Barach, Rami Ahmed

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Over 115,000 Health Care Workers (HCWs) have died from COVID-19, and millions have been infected while caring for patients. HCWs have filed thousands of safety complaints surrounding safety concerns due to Personal Protective Equipment (PPE) shortages, which included concerns around inadequate and PPE reuse. Protocols for donning and doffing PPE remain ambiguous, lacking an evidence-base, and often result in wide deviations in practice. PPE donning and doffing protocol deviations commonly result in self-contamination but have not been thoroughly addressed. No evidence-driven protocols provide guidance on protecting HCW during periods of PPE reuse. Objective: The aim of this study was to examine safety-related threats and risks to Health Care Workers (HCWs) due to the reuse of PPE among Emergency Department personnel. Method: We conducted a prospective observational study to examine the risks of reusing PPE. First, ED personnel were asked to don and doff PPE in a simulation lab. Each participant was asked to don and doff PPE five times, according to the maximum reuse recommendation set by the Centers for Disease Control and Prevention (CDC). Each participant was videorecorded; video recordings were reviewed and coded independently by at least 2 of the 3trained coders for safety behaviors and riskiness of actions. A third coder was brought in when the agreement between the 2 coders could not be reached. Agreement between coders was high (81.9%), and all disagreements (100%) were resolved via consensus. A bowtie risk assessment chart was constructed analyzing the factors that contribute to increased risks HCW are faced with due to PPE use and reuse. Agreement amongst content experts in the field of Emergency Medicine, Human Factors, and Anesthesiology was used to select aspects of health care that both contribute and mitigate risks associated with PPE reuse. Findings: Twenty-eight clinician participants completed five rounds of donning/doffing PPE, yielding 140 PPE donning/doffing sequences. Two emerging threats were associated with behaviors in donning, doffing, and re-using PPE: (i) direct exposure to contaminant, and (ii) transmission/spread of contaminant. Protective behaviors included: hand hygiene, not touching the patient-facing surface of PPE, and ensuring a proper fit and closure of all PPE materials. 100% of participants (n= 28) deviated from the CDC recommended order, and most participants (92.85%, n=26) self-contaminated at least once during reuse. Other frequent errors included failure to tie all ties on the PPE (92.85%, n=26) and failure to wash hands after a contamination event occurred (39.28%, n=11). Conclusions: There is wide variation and regular errors in how HCW don and doffPPE while including in reusing PPE that led to self-contamination. Some errors were deemed “recoverable”, such as hand washing after touching a patient-facing surface to remove the contaminant. Other errors, such as using a contaminated mask and accidentally spreading to the neck and face, can lead to compound risks that are unique to repeated PPE use. A more comprehensive understanding of the contributing threats to HCW safety and complete approach to mitigating underlying risks, including visualizing with risk management toolsmay, aid future PPE designand workflow and space solutions.

Keywords: bowtie analysis, health care, PPE reuse, risk management

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Authors: Tamar Lin, Nufar Buchshtab, Yifat Elharar, Julian Nicenboim, Rotem Edgar, Iddo Weiner, Lior Zelcbuch, Ariel Cohen, Sharon Kredo-Russo, Inbar Gahali-Sass, Naomi Zak, Sailaja Puttagunta, Merav Bassan

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Background: Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disorder that is characterized by dry skin and flares of eczematous lesions and intense pruritus. Multiple lines of evidence suggest that AD is associated with increased colonization by Staphylococcus aureus, which contributes to disease pathogenesis through the release of virulence factors that affect both keratinocytes and immune cells, leading to disruption of the skin barrier and immune cell dysfunction. The aim of the current study is to develop a bacteriophage-based product that specifically targets S. aureus. Methods: For the discovery of phage, environmental samples were screened on 118 S. aureus strains isolated from skin samples, followed by multiple enrichment steps. Natural phages were isolated, subjected to Next-generation Sequencing (NGS), and analyzed using proprietary bioinformatics tools for undesirable genes (toxins, antibiotic resistance genes, lysogeny potential), taxonomic classification, and purity. Phage host range was determined by an efficiency of plating (EOP) value above 0.1 and the ability of the cocktail to completely lyse liquid bacterial culture under different growth conditions (e.g., temperature, bacterial stage). Results: Sequencing analysis demonstrated that the 118 S. aureus clinical strains were distributed across the phylogenetic tree of all available Refseq S. aureus (~10,750 strains). Screening environmental samples on the S. aureus isolates resulted in the isolation of 50 lytic phages from different genera, including Silviavirus, Kayvirus, Podoviridae, and a novel unidentified phage. NGS sequencing confirmed the absence of toxic elements in the phages’ genomes. The host range of the individual phages, as measured by the efficiency of plating (EOP), ranged between 41% (48/118) to 79% (93/118). Host range studies in liquid culture revealed that a subset of the phages can infect a broad range of S. aureus strains in different metabolic states, including stationary state. Combining the single-phage EOP results of selected phages resulted in a broad host range cocktail which infected 92% (109/118) of the strains. When tested in vitro in a liquid infection assay, clearance was achieved in 87% (103/118) of the strains, with no evidence of phage resistance throughout the study (24 hours). A S. aureus host was identified that can be used for the production of all the phages in the cocktail at high titers suitable for large-scale manufacturing. This host was validated for the absence of contaminating prophages using advanced NGS methods combined with multiple production cycles. The phages are produced under optimized scale-up conditions and are being used for the development of a topical formulation (BX005) that may be administered to subjects with atopic dermatitis. Conclusions: A cocktail of natural phages targeting S. aureus was effective in reducing bacterial burden across multiple assays. Phage products may offer safe and effective steroid-sparing options for atopic dermatitis.

Keywords: atopic dermatitis, bacteriophage cocktail, host range, Staphylococcus aureus

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Authors: Elvin P. Chizenga, Heidi Abrahamse

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Cancer cell resistance to therapy is the main cause of treatment failures and the poor prognosis of cancer convalescence. The progression of cervical cancer to other parts of the genitourinary system and the reported recurrence rates are overwhelming. Current treatments, including surgery, chemo and radiation have been inefficient in eradicating the tumor cells. These treatments are also associated with poor prognosis and reduced quality of life, including fertility loss. This has inspired the need for the development of new treatment modalities to eradicate cervical cancer successfully. Photodynamic Therapy (PDT) is a modern treatment modality that induces cell death by photochemical interactions of light and a photosensitizer, which in the presence of molecular oxygen, yields a set of chemical reactions that generate Reactive Oxygen Species (ROS) and other free radical species causing cell damage. Enhancing PDT using modified drug delivery can increase the concentration of the photosensitizer in the tumor cells, and this has the potential to maximize its therapeutic efficacy. In cervical cancer, all infected cells constitutively express genes of the E6 and E7 HPV viral oncoproteins, resulting in high concentrations of E6 and E7 in the cytoplasm. This provides an opportunity for active targeting of cervical cancer cells using immune-mediated drug delivery to maximize therapeutic efficacy. The use of nanoparticles in PDT has also proven effective in enhancing therapeutic efficacy. Gold nanoparticles (AuNps) in particular, are explored for their use in biomedicine due to their biocompatibility, low toxicity, and enhancement of drug uptake by tumor cells. In this present study, a biomolecule comprising of AuNPs, anti-E6 monoclonal antibodies, and Aluminium Phthalocyanine photosensitizer was synthesized for use in targeted PDT of cervical cancer. The AuNp-Anti-E6-Sulfonated Aluminium Phthalocyanine mix (AlPcSmix) photosensitizing biomolecule was synthesized by coupling AuNps and anti-E6 monoclonal antibodies to the AlPcSmix via Polyethylene Glycol (PEG) chemical links. The final product was characterized using Transmission Electron Microscope (TEM), Zeta Potential, Uv-Vis Spectrophotometry, Fourier Transform Infrared Spectroscopy (FTIR), and X-ray diffraction (XRD), to confirm its chemical structure and functionality. To observe its therapeutic role in treating cervical cancer, cervical cancer cells, HeLa cells were seeded in 3.4 cm² diameter culture dishes at a concentration of 5x10⁵ cells/ml, in vitro. The cells were treated with varying concentrations of the photosensitizing biomolecule and irradiated using a 673.2 nm wavelength of laser light. Post irradiation cellular responses were performed to observe changes in morphology, viability, proliferation, cytotoxicity, and cell death pathways induced. Dose-Dependent response of the cells to treatment was demonstrated as significant morphologic changes, increased cytotoxicity, and decreased cell viability and proliferation This study presented a synthetic biomolecule for targeted PDT of cervical cancer. The study suggested that PDT using this AuNp- Anti-E6- AlPcSmix photosensitizing biomolecule is a very effective treatment method for the eradication of cervical cancer cells, in vitro. Further studies in vivo need to be conducted to support the use of this biomolecule in treating cervical cancer in clinical settings.

Keywords: anti-E6 monoclonal antibody, cervical cancer, gold nanoparticles, photodynamic therapy

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Authors: Michal Kielbik, Izabela Szulc-Kielbik, Anna Brzostek, Jaroslaw Dziadek, Magdalena Klink

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The goal of many research groups in the world is to find new components that are important for survival of mycobacteria in the host cells. Mycobacterium tuberculosis (Mtb) possesses a number of enzymes degrading cholesterol that are considered to be an important factor for its survival and persistence in host macrophages. One of them - cholesterol oxidase (ChoD), although not being essential for cholesterol degradation, is discussed as a virulence compound, however its involvement in macrophages’ response to Mtb is still not sufficiently determined. The recognition of tubercle bacilli antigens by pathogen recognition receptors is crucial for the initiation of the host innate immune response. An important receptor that has been implicated in the recognition and/or uptake of Mtb is Toll-like receptor type 2 (TLR2). Engagement of TLR2 results in the activation and phosphorylation of intracellular signaling proteins including IRAK-1 and -4, TRAF-6, which in turn leads to the activation of target kinases and transcription factors responsible for bactericidal and pro-inflammatory response of macrophages. The aim of these studies was a detailed clarification of the role of Mtb cholesterol oxidase as a virulence factor affecting the TLR2 signaling pathway in human macrophages. As human macrophages the THP-1 differentiated cells were applied. The virulent wild-type Mtb strain (H37Rv), its mutant lacking a functional copy of gene encoding cholesterol oxidase (∆choD), as well as complimented strain (∆choD–choD) were used. We tested the impact of Mtb strains on the expression of TLR2-depended signaling proteins (mRNA level, cytosolic level and phosphorylation status). The cytokine and bactericidal response of THP-1 derived macrophages infected with Mtb strains in relation to TLR2 signaling pathway dependence was also determined. We found that during the 24-hours of infection process the wild-type and complemented Mtb significantly reduced the cytosolic level and phosphorylation status of IRAK-4 and TRAF-6 proteins in macrophages, that was not observed in the case of ΔchoD mutant. Decreasement of TLR2-dependent signaling proteins, induced by wild-type Mtb, was not dependent on the activity of proteasome. Blocking of TLR2 expression, before infection, effectively prevented the induced by wild-type strain reduction of cytosolic level and phosphorylation of IRAK-4. None of the strains affected the surface expression of TLR2. The mRNA level of IRAK-4 and TRAF-6 genes were significantly increased in macrophages 24 hours post-infection with either of tested strains. However, the impact of wild-type Mtb strain on both examined genes was significantly stronger than its ΔchoD mutant. We also found that wild-type strain stimulated macrophages to release high amount of immunosuppressive IL-10, accompanied by low amount of pro-inflammatory IL-8 and bactericidal nitric oxide in comparison to mutant lacking cholesterol oxidase. The influence of wild-type Mtb on this type of macrophages' response strongly dependent on fully active IRAK-1 and IRAK-4 signaling proteins. In conclusion, Mtb using cholesterol oxidase causes the over-activation of TLR2 signaling proteins leading to the reduction of their cytosolic level and activity resulting in the modulation of macrophages response to allow its intracellular survival. Supported by grant: 2014/15/B/NZ6/01565, National Science Center, Poland

Keywords: Mycobacterium tuberculosis, cholesterol oxidase, macrophages, TLR2-dependent signaling pathway

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Authors: Seyedeh Nasim Mirbahari, Taha Azad, Mehdi Totonchi

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Oncolytic viruses, which only replicate in tumor cells, are being extensively studied for their use in cancer therapy. A particular virus known as the vaccinia virus, a member of the poxvirus family, has demonstrated oncolytic abilities glioma. Treating Glioma with traditional methods such as chemotherapy and radiotherapy is quite challenging. Even though oncolytic viruses have shown immense potential in cancer treatment, their effectiveness in glioblastoma treatment is still low. Therefore, there is a need to improve and optimize immunotherapies for better results. In this study, we have designed oncoVV-TT, which can more effectively target tumor cells while minimizing replication in normal cells by replacing the thymidine kinase gene with a luc-p2a-GFP gene expression cassette. Human glioblastoma cell line U251 MG, rat glioblastoma cell line C6, and non-tumor cell line HFF were plated at 105 cells in a 12-well plates in 2 mL of DMEM-F2 medium with 10% FBS added to each well. Then incubated at 37°C. After 16 hours, the cells were treated with oncoVV-TT at an MOI of 0.01, 0.1 and left in the incubator for a further 24, 48, 72 and 96 hours. Viral replication assay, fluorescence imaging and viability tests, including trypan blue and crystal violet, were conducted to evaluate the cytotoxic effect of oncoVV-TT. The finding shows that oncoVV-TT had significantly higher cytotoxic activity and proliferation rates in tumor cells in a dose and time-dependent manner, with the strongest effect observed in U251 MG. To conclude, oncoVV-TT has the potential to be a promising oncolytic virus for cancer treatment, with a more cytotoxic effect in human glioblastoma cells versus rat glioma cells. To assess the effectiveness of vaccinia virus-mediated viral therapy, we have tested U251mg and C6 tumor cell lines taken from human and rat gliomas, respectively. The study evaluated oncoVV-TT's ability to replicate and lyse cells and analyzed the survival rates of the tested cell lines when treated with different doses of oncoVV-TT. Additionally, we compared the sensitivity of human and mouse glioma cell lines to the oncolytic vaccinia virus. All experiments regarding viruses were conducted under biosafety level 2. We engineered a Vaccinia-based oncolytic virus called oncoVV-TT to replicate specifically in tumor cells. To propagate the oncoVV-TT virus, HeLa cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 10 MOI virus was added. After 48 h, cells were harvested by scraping, and viruses were collected by 3 sequential freezing and thawing cycles followed by removal of cell debris by centrifugation (1500 rpm, 5 min). The supernatant was stored at −80 ◦C for the following experiments. To measure the replication of the virus in Hela, cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 5 MOI virus or equal dilution of PBS was added. At the treatment time of 0 h, 24 h, 48 h, 72 h and 96 h, the viral titers were determined under the fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). Fluorescence intensity was quantified using the imagej software according to the manufacturer’s protocol. For the isolation of single-virus clones, HeLa cells seeded in six-well plates (5×105 cells/well). After 24 h (100% confluent), the cells were infected with a 10-fold dilution series of TianTan green fluorescent protein (GFP)virus and incubated for 4 h. To examine the cytotoxic effect of oncoVV-TT virus ofn U251mg and C6 cell, trypan blue and crystal violet assay was used.

Keywords: oncolytic virus, immune therapy, glioma, vaccinia virus

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Authors: Hosein I. Hosein, Ragab Azzam, Ahmed M. S. Menshawy, Sherin Rouby, Khaled Hendy, Ayman Mahrous, Hany Hussien

Abstract:

Brucellosis is a highly contagious bacterial zoonotic disease of a worldwide spread and has different names; Infectious or enzootic abortion and Bang's disease in animals; and Mediterranean or Malta fever, Undulant Fever and Rock fever in humans. It is caused by the different species of genus Brucella which is a Gram-negative, aerobic, non-spore forming, facultative intracellular bacterium. Brucella affects a wide range of mammals including bovines, small ruminants, pigs, equines, rodents, marine mammals as well as human resulting in serious economic losses in animal populations. In human, Brucella causes a severe illness representing a great public health problem. The disease was reported in Egypt for the first time in 1939; since then the disease remained endemic at high levels among cattle, buffalo, sheep and goat and is still representing a public health hazard. The annual economic losses due to brucellosis were estimated to be about 60 million Egyptian pounds yearly, but actual estimates are still missing despite almost 30 years of implementation of the Egyptian control programme. Despite being the gold standard, bacterial isolation has been reported to show poor sensitivity for samples with low-level of Brucella and is impractical for regular screening of large populations. Thus, serological tests still remain the corner stone for routine diagnosis of brucellosis, especially in developing countries. In the present study, a total of 1533 cows (256 from Beni-Suef Governorate, 445 from Al-Fayoum Governorate and 832 from Damietta Governorate), were employed for estimation of relative sensitivity, relative specificity, positive predictive value and negative predictive value of buffered acidified plate antigen test (BPAT), rose bengal test (RBT) and complement fixation test (CFT). The overall seroprevalence of brucellosis revealed (19.63%). Relative sensitivity, relative specificity, positive predictive value and negative predictive value of BPAT,RBT and CFT were estimated as, (96.27 %, 96.76 %, 87.65 % and 99.10 %), (93.42 %, 96.27 %, 90.16 % and 98.35%) and (89.30 %, 98.60 %, 94.35 %and 97.24 %) respectively. BPAT showed the highest sensitivity among the three employed serological tests. RBT was less specific than BPAT. CFT showed the least sensitivity 89.30 % among the three employed serological tests but showed the highest specificity. Different tissues specimens of 22 seropositive cows (spleen, retropharyngeal udder, and supra-mammary lymph nodes) were subjected for bacteriological studies for isolation and identification of Brucella organisms. Brucella melitensis biovar 3 could be recovered from 12 (54.55%) cows. Bacteriological examinations failed to classify 10 cases (45.45%) and were culture negative. Bruce-ladder PCR was carried out for molecular identification of the 12 Brucella isolates at the species level. Three fragments of 587 bp, 1071 bp and 1682 bp sizes were amplified indicating Brucella melitensis. The results indicated the importance of using several procedures to overcome the problem of escaping of some infected animals from diagnosis.Bruce-ladder PCR is an important tool for diagnosis and epidemiologic studies, providing relevant information for identification of Brucella spp.

Keywords: brucellosis, relative sensitivity, relative specificity, Bruce-ladder, Egypt

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Authors: Anushka Naidoo, Veron Ramsuran, Maxwell Chirehwa, Paolo Denti, Kogieleum Naidoo, Helen McIlleron, Nonhlanhla Yende-Zuma, Ravesh Singh, Sinaye Ngcapu, Nesri Padayatachi

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Background: Despite efforts to introduce new drugs and shorter drug regimens for drug-susceptible tuberculosis (TB), the standard first-line treatment has not changed in over 50 years. Rifampicin, isoniazid, and pyrazinamide are critical components of the current standard treatment regimens. Some studies suggest that microbiologic failure and acquired drug resistance are primarily driven by low drug concentrations that result from pharmacokinetic (PK) variability independent of adherence to treatment. Wide between-patient pharmacokinetic variability for rifampin, isoniazid, and pyrazinamide has been reported in prior studies. There may be several reasons for this variability. However, genetic variability in genes coding for drug metabolizing and transporter enzymes have been shown to be a contributing factor for variable tuberculosis drug exposures. Objective: We describe the pharmacokinetics of first-line TB drugs rifampicin, isoniazid, and pyrazinamide and assess the effect of genetic variability in relevant selected drug metabolizing and transporter enzymes on pharmacokinetic parameters of isoniazid and rifampicin. Methods: We conducted the randomized-controlled Improving retreatment success TB trial in Durban, South Africa. The drug regimen included rifampicin, isoniazid, and pyrazinamide. Drug concentrations were measured in plasma, and concentration-time data were analysed using nonlinear-mixed-effects models to quantify the effects of relevant covariates and single nucleotide polymorphisms (SNP’s) of drug metabolizing and transporter genes on rifampicin, isoniazid and pyrazinamide exposure. A total of 25 SNP’s: four NAT2 (used to determine acetylator status), four SLCO1B1, three Pregnane X receptor (NR1), six ABCB1 and eight UGT1A, were selected for analysis in this study. Genotypes were determined for each of the SNP’s using a TaqMan® Genotyping OpenArray™. Results: Among fifty-eight patients studied; 41 (70.7%) were male, 97% black African, 42 (72.4%) HIV co-infected and 40 (95%) on efavirenz-based ART. Median weight, fat-free mass (FFM), and age at baseline were 56.9 kg (interquartile range, IQR: 51.1-65.2), 46.8 kg (IQR: 42.5-50.3) and 37 years (IQR: 31-42), respectively. The pharmacokinetics of rifampicin and pyrazinamide was best described using one-compartment models with first-order absorption and elimination, while for isoniazid two-compartment disposition was used. The median (interquartile range: IQR) AUC (h·mg/L) and Cmax (mg/L) for rifampicin, isoniazid, and pyrazinamide were; 25.62 (23.01-28.53) and 4.85 (4.36-5.40), 10.62 (9.20-12.25) and 2.79 (2.61-2.97), 345.74 (312.03-383.10) and 28.06 (25.01-31.52), respectively. Eighteen percent of patients were classified as rapid acetylators, and 34% and 43% as slow and intermediate acetylators, respectively. Rapid and intermediate acetylator status based on NAT 2 genotype resulted in 2.3 and 1.6 times higher isoniazid clearance than slow acetylators. We found no effects of the SLCO1B1 genotypes on rifampicin pharmacokinetics. Conclusion: Plasma concentrations of rifampicin, isoniazid, and pyrazinamide were low overall in our patients. Isoniazid clearance was high overall and as expected higher in rapid and intermediate acetylators resulting in lower drug exposures. In contrast to reports from previous South African or Ugandan studies, we did not find any effects of the SLCO1B1 or other genotypes tested on rifampicin PK. However, our findings are in keeping with more recent studies from Malawi and India emphasizing the need for geographically diverse and adequately powered studies. The clinical relevance of the low tuberculosis drug concentrations warrants further investigation.

Keywords: rifampicin, isoniazid pharmacokinetics, genetics, NAT2, SLCO1B1, tuberculosis

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Authors: Ikele Chika Bright, Mgbenka Bernard Obialo, Ikele Chioma Faith

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Icthyophthiriasis is a prevalent protozoan (ectoparasite) mostly affecting cultured and aquarium fishes. The majority of the chemotherapeutants lack efficacy for completely eliminating Ich parasite without affecting the environment and they are not safe for human health. The present work is focused on the evaluating different immersion treatments of African catfish (Clarias gariepinus) infected with ichthyophthiriasis and treated with a non-chemical and environmental friendly parasiticides Moringa oleifera. A total number of 800 apparently healthy parasites free (examined) post juvenile catfish were obtained from a reputable farm, disinfected with potassium permanganate in a quarantine tank to remove any possible external parasites. The fish were further challenged with approximately 44,000 infective stages of theronts which were obtained through serial passages by cohabitation. Seven groups (A-G) of post Juvenile were used for the experiment which was carried out into three stages; Dips (60minutes), short term treatment (24-96h) and prolong bath treatment (0-15 days). The concentrations selected were dependent on the outcome of the LC50 of the plant material from which dose-dependent factors were used to select various concentrations of the treatment. In Dips treatment, group D-G were treated with 1,500mg/L, 2500mg/L., 3500mg/L and 4500mg/L, short-term treatment was treated with 150mg/L, 250mg/L, 350mg/L and 450mg/L and prolong bath was treated with 15mg/L, 25mg/L, 35mg/L and 45mg/L of the plant extract whereas group A, B and C were normal control, Ich- infested not treated and Ich- infested treated with standard drug (Acriflavin), respectively. The various types of treatment applied with corresponding concentrations showed almost complete elimination of the adult parasites (trophonts) both in the gills and the body smear, thereby making M. oleifera a potential parasiticides. There were serious pathological alterations in the skin and gills which are usually the main point for Ich parasites invasion but no significant morphological characteristics was noted among the treated groups subjected to different immersion treatment patterns. Epitheliocystis, aneurysm, oedema, hemorrhage, and localization of the adult parasite in the gills were the overall common observations made in the gills whereas degeneration of muscle fibre, dermatitis, hemorrhage, oedema, abscess formation and keratinisation were observed in the skin. However, there are no pathological changes in the control group. Moreover, biochemical parameters such as urea, creatinine, albumin., globulin, total protein, ALT, AST), blood chemistry (sodium, chloride, potassium, bicarbonate), antioxidants (CAT, SOD, GPx, LPO), enzymatic activities (myeloperoxidase, thioreadoxin reductase), Inflammatory response (C-reactive protein), Stress markers (lactate dehydrogenase), heamatological parameters (RBC, PCV, WBC, HB and differential count), lipid profile (total cholesterol, tryglycerides , high density lipoprotein and low density lipoprotein) all showed various significant (P<0.05) and no significant (P>0.05) responses among the Ich-infested fish treated under three immersion treatments. It is suggested that M. oleifera may serve as an alternatives to chemotherapeutants for control of Ichthyophthiriasis in African catfish Clarias gariepinus.

Keywords: Icthyophthirius multifilis, immersion treatment, pathophysiology, African catfish

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Authors: Asim Abbasi, Muhammad Sufyan, Iqra, Hafiza Javaria Ashraf

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The share of vector-borne diseases (VBDs) in the global burden of infectious diseases is almost 17%. The advent of new drugs and latest research in medical science helped mankind to compete with these lethal diseases but still diseases transmitted by different mosquito species, including filariasis, malaria, viral encephalitis and dengue are serious threats for people living in disease endemic areas. Injudicious and repeated use of pesticides posed selection pressure on mosquitoes leading to development of resistance. Hence biological control agents are under serious consideration of scientific community to be used in vector control programmes. Fish have a history of predating immature stages of different aquatic insects including mosquitoes. The noteworthy examples in Africa and Asia includes, Aphanius discolour and a fish in the Panchax group. Moreover, common mosquito fish, Gambusia affinis predates mostly on temporary water mosquitoes like anopheline as compared to permanent water breeders like culicines. Mosquitoes belonging to genus Toxorhynchites have a worldwide distribution and are mostly associated with the predation of other mosquito larvae habituating with them in natural and artificial water containers. These species are harmless to humans as their adults do not suck human blood but feeds on floral nectar. However, their activity is mostly temperature dependent as Toxorhynchites brevipalpis consume 359 Aedes aegypti larvae at 30-32 ºC in contrast to 154 larvae at 20-26 ºC. Although many bacterial species were isolated from mosquito cadavers but those belonging to genus Bacillus are found highly pathogenic against them. The successful species of this genus include Bacillus thuringiensis and Bacillus sphaericus. The prime targets of B. thuringiensis are mostly the immatures of genus Aedes, Culex, Anopheles and Psorophora while B. sphaericus is specifically toxic against species of Culex, Psorophora and Culiseta. The entomopathogenic nematodes belonging to family, mermithidae are also pathogenic to different mosquito species. Eighty different species of mosquitoes including Anopheles, Aedes and Culex proved to be highly vulnerable to the attack of two mermithid species, Romanomermis culicivorax and R. iyengari. Cytoplasmic polyhedrosis virus was the first described pathogenic virus, isolated from the cadavers of mosquito specie, Culex tarsalis. Other viruses which are pathogenic to culicine includes, iridoviruses, cytopolyhedrosis viruses, entomopoxviruses and parvoviruses. Protozoa species belonging to division microsporidia are the common pathogenic protozoans in mosquito populations which kill their host by the chronic effects of parasitism. Moreover, due to their wide prevalence in anopheline mosquitoes and transversal and horizontal transmission from infected to healthy host, microsporidia of the genera Nosema and Amblyospora have received much attention in various mosquito control programmes. Fungal based mycopesticides are used in biological control of insect pests with 47 species reported virulent against different stages of mosquitoes. These include both aquatic fungi i.e. species of Coelomomyces, Lagenidium giganteum and Culicinomyces clavosporus, and the terrestrial fungi Metarhizium anisopliae and Beauveria bassiana. Hence, it was concluded that the integrated use of all these biological control agents can be a healthy contribution in mosquito control programmes and become a dire need of the time to avoid repeated use of pesticides.

Keywords: entomopathogenic nematodes, protozoa, Toxorhynchites, vector-borne

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Authors: Jayanwita Sarkar, Usha Chakraborty, Bishwanath Chakraborty

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Plants are constantly interacting with various abiotic and biotic stresses. In changing climate scenario plants are continuously modifying physiological processes to adapt to changing environmental conditions which profoundly affect plant-pathogen interactions. Spot blotch in wheat is a fast-rising disease in the warmer plains of South Asia where the rise in minimum average temperature over most of the year already affecting wheat production. Hence, the study was undertaken to explore the role of elevated temperature in spot blotch disease development and modulation of antioxidative responses by plant growth promoting rhizobacteria (PGPR) for biocontrol of spot blotch at high temperature. Elevated temperature significantly increases the susceptibility of wheat plants to spot blotch causing pathogen Bipolaris sorokiniana. Two PGPR Bacillus safensis (W10) and Ochrobactrum pseudogrignonense (IP8) isolated from wheat (Triticum aestivum L.) and blady grass (Imperata cylindrical L.) rhizophere respectively, showing in vitro antagonistic activity against Bipolaris sorokiniana were tested for growth promotion and induction of resistance against spot blotch in wheat. GC-MS analysis showed that Bacillus safensis (W10) and Ochrobactrum pseudogrignonense (IP8) produced antifungal and antimicrobial compounds in culture. Seed priming with these two bacteria significantly increase growth, modulate antioxidative signaling and induce resistance and eventually reduce disease incidence in wheat plants at optimum as well as elevated temperature which was further confirmed by indirect immunofluorescence assay using polyclonal antibody raised against Bipolaris sorokiniana. Application of the PGPR led to enhancement in activities of plant defense enzymes- phenylalanine ammonia lyase, peroxidase, chitinase and β-1,3 glucanase in infected leaves. Immunolocalization of chitinase and β-1,3 glucanase in PGPR primed and pathogen inoculated leaf tissue was further confirmed by transmission electron microscopy using PAb of chitinase, β-1,3 glucanase and gold labelled conjugates. Activity of ascorbate-glutathione redox cycle related enzymes such as ascorbate peroxidase, superoxide dismutase and glutathione reductase along with antioxidants such as carotenoids, glutathione and ascorbate and osmolytes like proline and glycine betain accumulation were also increased during disease development in PGPR primed plant in comparison to unprimed plants at high temperature. Real-time PCR analysis revealed enhanced expression of defense genes- chalcone synthase and phenyl alanineammonia lyase. Over expression of heat shock proteins like HSP 70, small HSP 26.3 and heat shock factor HsfA3 in PGPR primed plants effectively protect plants against spot blotch infection at elevated temperature as compared with control plants. Our results revealed dynamic biochemical cross talk between elevated temperature and spot blotch disease development and furthermore highlight PGPR mediated array of antioxidative and molecular alterations responsible for induction of resistance against spot blotch disease at elevated temperature which seems to be associated with up-regulation of defense genes, heat shock proteins and heat shock factors, less ROS production, membrane damage, increased expression of redox enzymes and accumulation of osmolytes and antioxidants.

Keywords: antioxidative enzymes, defense enzymes, elevated temperature, heat shock proteins, PGPR, Real-Time PCR, spot blotch, wheat

Procedia PDF Downloads 156

Authors: Federica Verna, Alessandra Pautasso, Maria Caramelli, Cristiana Maurella, Walter Mignone, Cristina Casalone

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Mosquito-Borne Diseases (MBDs) are dangerously increasing in prevalence, geographical distribution and severity, representing an emerging threat for both humans and animals. Interaction between multiple disciplines is needed for an effective early warning, surveillance and control of MBDs, according to the One Health concept. This work reports the integrated surveillance system enforced by IZSPLV in Piedmont, Liguria and Valle d’Aosta regions (Northwestern Italy) in order to control MDBs spread. Veterinary services and local human health authority are involved in an information network, to connect the surveillance of human clinical cases with entomological surveillance and veterinary monitoring in order to implement control measures in case of outbreak. A systematic entomological surveillance is carried out during the vector season using mosquitoes traps located in sites selected according to risk factors. Collected mosquitoes are counted, identified to species level by morphological standard classification keys and pooled by collection site, date and species with a maximum of 100 individuals. Pools are analyzed, after RNA extraction, by Real Time RT-PCR distinctive for West Nile Virus (WNV) Lineage 1 and Lineage 2, Real Time RT-PCR USUTU virus (USUV) and a traditional flavivirus End-point RT-PCR. Positive pools are sequenced and the related sequences employed to perform a basic local alignment search tool (BLAST) in the GenBank library. Positive samples are sent to the National Reference Centre for Animal Exotic Diseases (CESME, Teramo) for confirmation. With particular reference to WNV, after the confirmation, as provided by national legislation, control measures involving both local veterinary and human health services are activated: equine sera are randomly sampled within a 4 km radius from the positive collection sites and tested with ELISA kit and WNV NAT screening of blood donors is introduced. This surveillance network allowed to detect since 2011 USUV circulation in this area of Italy. WNV was detected in Piedmont and Liguria for the first time in 2014 in mosquitoes. During the 2015 vector season, we observed the expansion of its activity in Piedmont. The virus was detected in almost all Provinces both in mosquitoes (6 pools) and animals (19 equine sera, 4 birds). No blood bag tested resulted infected. The first neuroinvasive human case occurred too. Competent authorities should be aware of a potentially increased risk of MBDs activity during the 2016 vector season. This work shows that this surveillance network allowed to early detect the presence of MBDs in humans and animals, and provided useful information to public authorities, in order to apply control measures. Finally, an additional value of our diagnostic protocol is the ability to detect all viruses belonging to the Flaviviridae family, considering the emergence caused by other Flaviviruses in humans such as the recent Zika virus infection in South America. Italy has climatic and environmental features conducive to Zika virus transmission, the competent vector and many travellers from Brazil reported every year.

Keywords: integrated surveillance, mosquito borne disease, West Nile virus, Zika virus

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Authors: Patricia Shirley Almeida Prado, Selma Paixão Argollo, Maria De Fátima Teixeira Guimarães, Leticia Matos Sobrinho

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Skeletal differential diagnosis is essential in forensic anthropology in order to differentiate skeletal trauma from normal osseous variation and pathological processes. Thus, part of forensic anthropological field is differentiate skeletal criminal injuries from the normal skeletal variation (bone fusion or nonunion, transitional vertebrae and other non-metric traits), non-traumatic skeletal pathology (myositis ossificans, arthritis, bone metastasis, osteomyelitis) from traumatic skeletal pathology (myositis ossificans traumatic) avoiding misdiagnosis. This case shows the importance of effective pathological diagnosis in order to accelerate the identification process of skeletonized human remains. THE CASE: An unidentified skeletal remains at the medico legal institute Nina Rodrigues-Salvador, of a male young adult (29 to 40 years estimated) showing a massive heterotopic ossification on its right tibia at upper epiphysis and adjacent articular femur surface; an extensive ossification on the right clavicle (at the sternal extremity) also presenting an heterotopic ossification at right scapulae (upper third of scapulae lateral margin and infraglenoid tubercule) and at the head of right humerus at the shoulder joint area. Curiously, this case also shows an unusual porosity in certain vertebrae´s body and in some tarsal and carpal bones. Likewise, his left fifth metacarpal bones (right and left) showed a healed fracture which led both bones distorted. Based on identification, of pathological conditions in human skeletal remains literature and protocols these alterations can be misdiagnosed and this skeleton may present more than one pathological process. The anthropological forensic lab at Medico-legal Institute Nina Rodrigues in Salvador (Brazil) adopts international protocols to ancestry, sex, age and stature estimations, also implemented well-established conventions to identify pathological disease and skeletal alterations. The most compatible diagnosis for this case is hematogenous osteomyelitis due to following findings: 1: the healed fracture pattern at the clavicle showing a cloaca which is a pathognomonic for osteomyelitis; 2: the metacarpals healed fracture does not present cloaca although they developed a periosteal formation. 3: the superior articular surface of the right tibia shows an extensive inflammatory healing process that extends to adjacent femur articular surface showing some cloaca at tibia bone disease. 4: the uncommon porosities may result from hematogenous infectious process. The fractures probably have occurred in a different moments based on the healing process; the tibia injury is more extensive and has not been reorganized, while metacarpals and clavicle fracture is properly healed. We suggest that the clavicle and tibia´s fractures were infected by an existing infectious disease (syphilis, tuberculosis, brucellosis) or an existing syndrome (Gorham’s disease), which led to the development of osteomyelitis. This hypothesis is supported by the fact that different bones are affected in diverse levels. Like the metacarpals that do not show the cloaca, but then a periosteal new bone formation; then the unusual porosities do not show a classical osteoarthritic processes findings as the marginal osteophyte, pitting and new bone formation, they just show an erosive process without bone formation or osteophyte. To confirm and prove our hypothesis we are working on different clinical approaches like DNA, histopathology and other image exams to find the correct diagnostic.

Keywords: bone disease, forensic anthropology, hematogenous osteomyelitis, human identification, human remains

Procedia PDF Downloads 317

Authors: Nino Kupatadze, Tamar Memanishvili, Natia Ochkhikidze, David Tugushi, Zaal Kokaia, Ramaz Katsarava

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Amino acid-based Biodegradable poly(ester-amide)s (PEAs) have gained considerable interest as a promising materials for numerous biomedical applications. These polymers reveal a high biocompatibility and easily form small particles suitable for delivery various biological, as well as elastic bio-erodible films serving as matrices for constructing antibacterial coatings. In the present work we have demonstrated a potential of the PEAs for two applications: 1. cell therapy for stroke as vehicles for delivery and sustained release of growth factors, 2. bactericidal coating as prevention biofilm and applicable in infected wound management. Stroke remains the main cause of adult disability with limited treatment options. Although stem cell therapy is a promising strategy, it still requires improvement of cell survival, differentiation and tissue modulation. .Recently, microspheres (MPs) made of biodegradable polymers have gained significant attention for providing necessary support of transplanted cells. To investigate this strategy in the cell therapy of stroke, MPs loaded with transcription factors Wnt3A/BMP4 were prepared. These proteins have been shown to mediate the maturation of the cortical neurons. We have suggested that implantation of these materials could create a suitable microenvironment for implanted cells. Particles with spherical shape, porous surface, and 5-40 m in size (monitored by scanning electron microscopy) were made on the basis of the original PEA composed of adipic acid, L-phenylalanine and 1,4-butanediol. After 4 months transplantation of MPs in rodent brain, no inflammation was observed. Additionally, factors were successfully released from MPs and affected neuronal cell differentiation in in vitro. The in vivo study using loaded MPs is in progress. Another severe problem in biomedicine is prevention of surgical devices from biofilm formation. Antimicrobial polymeric coatings are most effective “shields” to protect surfaces/devices from biofilm formation. Among matrices for constructing the coatings preference should be given to bio-erodible polymers. Such types of coatings will play a role of “unstable seating” that will not allow bacteria to occupy the surface. In other words, bio-erodible coatings would be discomfort shelter for bacteria that along with releasing “killers of bacteria” should prevent the formation of biofilm. For this purpose, we selected an original biodegradable PEA composed of L-leucine, 1,6-hexanediol and sebacic acid as a bio-erodible matrix, and nanosilver (AgNPs) as a bactericidal agent (“killer of bacteria”). Such nanocomposite material is also promising in treatment of superficial wound and ulcer. The solubility of the PEA in ethanol allows to reduce AgNO3 to NPs directly in the solution, where the solvent served as a reductive agent, and the PEA served as NPs stabilizer. The photochemical reduction was selected as a basic method to form NPs. The obtained AgNPs were characterized by UV-spectroscopy, transmission electron microscope (TEM), and dynamic light scattering (DLS). According to the UV-data and TEM data the photochemical reduction resulted in spherical AgNPs with wide particle size distribution with a high contribution of the particles below 10 nm that are known as responsible for bactericidal activity of AgNPs. DLS study showed that average size of nanoparticles formed after photo-reduction in ethanol solution ranged within ca. 50 nm.

Keywords: biodegradable polymers, microparticles, nanocomposites, stem cell therapy, stroke

Procedia PDF Downloads 385