A Unique Case of COVID-19 Presenting as Focal Seizures With Impaired Awareness.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has very rapidly become a global pandemic with millions of confirmed cases worldwide. In early 2021, viral encephalitis was the first neurological complication associated with COVID-19 and since then rise in cases has been reported with this association. A review highlighting 3 potential mechanisms linking a correlation between seizures and COVID-19 was previously reported. Herein described is a unique case of SARS-CoV2 infection that presented with focal seizure with impaired awareness.

A Unique Case of Pseudomonas aeruginosa-Associated Diarrhea in a Long-Term Hospitalized Adult Patient.

A 53-year-old Caucasian man with a history of alcohol use disorder, hypertension, and hypothyroidism presented with a myxedema coma requiring intubation. He had a complicated hospital course with ventilator-associated pneumonia with MRSA, sepsis with candida, and abdominal compartment syndrome requiring decompressive laparotomy. The patient slowly recovered during 43 days of hospitalization. During the intensive care unit (ICU) stay, a flexi-seal rectal tube was placed due to fecal incontinence. After being moved to a regular medicine unit, he started having loose watery stools with leukocytosis and neutrophilia. Clostridium difficile (C. diff.) colitis was suspected, and he was placed on oral vancomycin empirically. His stool test for C. diff. came back negative, and his rectal tube was subsequently removed. Imaging did not show any abscess, perforated viscus, or fistula formations. His stool culture grew a heavy colony of Pseudomonas aeruginosa (P. aeruginosa). Vancomycin was stopped, and he was started on oral ciprofloxacin 750 mg twice a day with complete resolution of his diarrhea and leukocytosis.

Malposition of the central venous catheter secondary to accessory hemiazygos vein variant.

A 36-year-old female with sickle cell disease presented with sickle cell pain crisis. After failure to establish peripheral venous access, an internal jugular central venous catheter (CVC) was placed. Confirmation of internal jugular cannulation was performed with bedside ultrasound. A confirmatory chest X-ray revealed an unusual position of the catheter, taking a course inferiorly, making a loop and remaining on the left side of the mediastinum. A lateral view was done and revealed that the catheter passed inferiorly through the internal jugular vein then posteriorly and inferiorly giving the looped appearance. This is better delineated on a sagittal view CT scan showing the tip of the catheter terminating in the accessory hemiazygos vein. This unusual course is due to a variant of the accessory hemiazygos vein which is connected to the left superior intercostal vein. This creates a lower resistance pathway for the CVC which passes from the internal jugular vein, down the left superior intercostal vein (instead of the left brachiocephalic vein) and into the accessory hemiazygos vein. Discussion: The correct tip placement of an internal jugular CVC terminates in the superior vena cava just above the cardiac silhouette. In 1%-2% of individuals, a connection between the accessory hemiazygos and the left superior intercostal vein is present. Rare cases are discovered incidentally during CVC placement. The diameter of the accessory hemiazygos vein is less than half of that of the superior vena cava. The catheter should not be used as central venous access and removal is recommended. Malpositioning of central catheters is unpredictable but can be easily avoided by using intraprocedural methods to confirm tip position. Such modalities include intracavitary ECG or ultrasound with agitated saline injection as described in the SIC (Safe Insertion of Centrally Inserted Central Catheters) protocol.

A Double Whammy Pneumonia: The First Reported Case of Concurrent Neisseria meningitidis and SARS-CoV-2 Pneumonia.

Meningococcal pneumonia (MP) is a rare manifestation of meningococcal disease. The MP was first described in 1907 when Neisseria meningitidis (NM) isolates were identified in sputum samples obtained from soldiers with pneumonia. Preceding and concurrent viral infections constitute a major risk for MP. During the 1918-1919 influenza pandemic, a significant increase in MP cases were reported in patients with preceding influenza infection. Despite the end of the last H1N1 influenza pandemic in 2010, seasonal influenza infections still pose a risk for simultaneous MP. History appears to be repeating itself with concomitant bacterial and viral coinfection amid the current SARS-CoV-2 pandemic. Herein presented is a unique case of an elderly woman who presented with, to the best of our knowledge, the first reported case of possible concurrent SARS-CoV-2 and MP infections.

HHV-8-Associated Multicentric Castleman Disease, a Diagnostic Challenge in a Patient With Acquired Immunodeficiency Syndrome and Fever.

Patients with acquired immunodeficiency syndrome (AIDS) are at an increased susceptibility to pathogens and associated malignancies which can present with a unique constellation of symptoms. In this article, we describe a case of Castleman disease in a patient with AIDS, nonadherent with antiretroviral therapy (ART), who presented with fevers, constant abdominal pain, nausea, and vomiting. After an extensive work up, a lymph node biopsy confirmed a diagnosis of human herpesvirus-8 (HHV-8)-associated multicentric Castleman disease. Patients presenting with AIDS and fever have broad differential diagnoses; therefore, reaching a diagnosis as rare as Castleman disease can be challenging. HHV-8 has a propensity to CD20 positive B cells, which allows rituximab to be an effect treatment.

Experiences and perception towards reproductive health education among secondary school teachers in South India.

BACKGROUND: Reproductive health education (RHE) is an important component of school curricula. It helps students in the decision-making process regarding several issues concerning reproductive health. However delivering RHE at schools is a difficult task for the teachers. METHODS: This study was conducted to assess the experiences and perceptions towards reproductive health education (RHE) among 236 secondary school teachers in January 2019. Data were collected using a self-administered questionnaire. RESULTS: Only 21 (8.9%) were trained in RHE. Majority [179 (75.8%)] identified cultural barriers as the major challenge involved in its implementation. 95 (40.3%) teachers felt that the provision of sexual education as a part of RHE will promote pre-marital sexual activity among the students. Of the total, 185 (78.4%) had average while 51 (21.6%) participants had a good perception towards RHE. It was taught in only 3 (16.7%) out of the 18 schools surveyed. Only 11 (4.7%) participants felt that the availability of teaching aids to conduct RHE classes at their schools was adequate. Hardly 14 (5.9%) teachers had taken RHE classes for students. Among the rest, 135 (60.8%) expressed their willingness to take RHE classes with appropriate training. In multi variable analysis, participants aged ≤ 40 years (p = 0.031), those belonging to nuclear families (p = 0.013), and those who had taken classes in RHE (p = 0.037) had significantly good perception level towards RHE. CONCLUSIONS: Teachers therefore need to be trained and given more opportunities to take RHE sessions which will help improve their perception towards RHE. Schools need to be better equipped with resources and various perceived barriers need to be overcome before RHE can be successfully implemented.

This study was conducted to assess the experiences and perceptions towards reproductive health education (RHE) secondary school teachers. The participants provided the required information by filling a questionnaire. Hardly one in ten of them had prior training in RHE and one in twenty had taken RHE classes at schools. More than three-fourth of them felt that cultural barriers could pose problems in its implementation at schools. One in four teachers had good perception towards RHE. Two in three among teachers, who had not taken RHE classes before, expressed their willingness to take RHE classes with appropriate training. Favourable perception towards RHE were expressed by teachers who were young, from small families and those who had taken RHE classes before.

Assessing Inflammatory Status of Pulp in Irreversible Pulpitis Cases with Pulse oximeter and Dental Hemogram.

Objective: This study evaluated pulse oximetry and dental hemogram in teeth with the clinical diagnosis of irreversible pulpitis (IP) to assess the inflammatory status of the pulp. Study design: The study and control groups (30n each) had teeth with IP and sound teeth respectively. Patients in the study group had night pain with or without pain on mastication (NM, N). Blood oxygen saturation (%SpO2) was recorded with a custom made pulse oximeter (CPO). For dental and peripheral hemogram, smears were made for each patient from the first drop of blood while entering the pulp and finger blood respectively. Results: Control group had mean %SpO2 in finger 91% (86-97); and in teeth 84% (80-91), while the study group had mean %SpO2 in finger 92% (88-98) and in teeth 83% (71-94). Fifty percent of IP cases were vital while no tooth showed necrosis according to CPO which was further confirmed by bleeding status from the pulp. Based on the findings of the clinical diagnosis, %SpO2 and bleeding status of IP and normal cases, the terminology as coronal or total pulpitis seems more appropriate. The statistical difference was significant in fingers while non-significant in teeth of IP and normal pulp cases. Dental hemogram of IP cases showed an overall significant fall of neutrophil, lymphocyte, eosinophil and monocyte counts compared to normal. Conclusion: Pulse oximetry was the most accurate pulp test to diagnose vitality in normal as well as inflamed pulps while hemogram was inconclusive for the same.

In Vitro and In Silico Studies of Glycyrrhetinic Acid Derivatives as Anti- Filarial Agents.

BACKGROUND: Lymphatic filariasis is one of the chronic diseases in many parts of the tropics and sub-tropics of the world despite the use of standard drugs diethylcarbamazine and ivermectin because they kill microfilaries and not the adult parasites. Therefore, new leads with activity on adult parasites are highly desirable. OBJECTIVE: Anti-filarial lead optimization by semi-synthetic modification of glycyrrhetinic acid (GA). METHODS: The GA was first converted into 3-O-acyl derivative, which was further converted into 12 amide derivatives. All these derivatives were assessed for their antifilarial potential by parasite motility assay. The binding affinity of active GA derivatives on trehalose-6-phosphate phosphatase (Bm-TPP) was assessed by molecular docking studies. RESULTS: Among 15 GA derivatives, GAD-2, GAD-3, and GAD-4 were found more potent than the GA and standard drug DEC. These derivatives reduced the motility of Brugia malayi adult worms by up to 74% while the GA and DEC reduced only up to 49%. Further, GA and most of its derivatives exhibited two times more reduction in MTT assay when compared to the standard drug DEC. These derivatives also showed 100% reduction of microfilariae and good interactions with Bm-TPP protein. CONCLUSION: The present study suggests that 3-O-acyl and linear chain amide derivatives of glycyrrhetinic acid may be potent leads against B. malayi microfilariae and adult worms. These results might be helpful in developing QSAR model for optimizing a new class of antifilarial lead from a very common, inexpensive, and non toxic natural product.

Severe Coagulopathy as a Rare Feature of Celiac Crisis in a Patient Previously Diagnosed with Celiac Disease.

BACKGROUND Celiac crisis is an uncommon but critical complication of celiac disease (CD) manifesting with copious diarrhea, dehydration, and severe metabolic imbalances. Celiac crisis occurring in individuals who have been formerly diagnosed with CD and displaying severe coagulopathy is tremendously rare. CASE REPORT We report a case of a 76-year-old male, previously diagnosed with CD and non-compliant with gluten free diet, who presented with severe coagulopathy manifesting as gastrointestinal bleeding in addition to other features of celiac crisis, including severe diarrhea, dehydration, metabolic acidosis, electrolyte disturbances, and renal dysfunction. Esophagogastroduodenoscopy revealed flattened mucosa and mucosal nodularity in the duodenum. Duodenal biopsies exhibited active chronic inflammation with intraepithelial lymphocytosis and subtotal villous blunting. The patient was diagnosed with celiac crisis and treatment with vitamin K, parenteral nutrition, and steroids was commenced. After initial clinical improvement, a gluten-free diet was implemented with complete resolution of symptoms. CONCLUSIONS Though celiac crisis typically presents in patients with undiagnosed CD, it should be considered in patients who have been previously diagnosed CD but who are non-compliant with gluten free diet. Severe coagulopathy, though extremely rare, can be a feature of celiac crisis and should be consider when encountered in a patient with history of steatorrhea and gastrointestinal bleeding.

Zebrafish model of amyloid light chain cardiotoxicity: regeneration versus degeneration.

Cardiac dysfunction is the most frequent cause of morbidity and mortality in amyloid light chain (AL) amyloidosis caused by a clonal immunoglobulin light chain (LC). Previously published transgenic animal models of AL amyloidosis have not recapitulated the key phenotype of cardiac dysfunction seen in AL amyloidosis, which has limited our understanding of the disease mechanisms in vivo, as well as the development of targeted AL therapeutics. We have developed a transgenic zebrafish model in which a λ LC derived from a patient with AL amyloidosis is conditionally expressed in the liver under the control of the Gal4 upstream activation sequence enhancer system. Circulating LC levels of 125 µg/ml in these transgenic zebrafish are comparable to median pathological serum LC levels. Functional analysis links abnormal contractile function with evidence of cellular and molecular proteotoxicity in the heart, including increased cell death and autophagy. However, despite pathological and functional phenotypes analogous to human AL, the lifespan of the transgenic fish is comparable to control fish without the expressed AL-LC transgene. Nuclear labeling experiments suggest increased cardiac proliferation in the transgenic fish, which can be counteracted by treatment with a small molecule proliferation inhibitor leading to increased zebrafish mortality because of cardiac apoptosis and functional deterioration. This transgenic zebrafish model provides a platform to study underlying AL disease mechanisms in vivo further. NEW & NOTEWORTHY Heart failure is a major cause of mortality in amyloid light (AL) amyloidosis, yet it has been difficult to model in animals. We report the generation of a transgenic zebrafish model for AL amyloidosis with pathological concentration of circulating human light chain protein that results in cardiac dysfunction. The light chain toxicity triggers regeneration in the zebrafish heart resulting in functional compensation early in life, but with age develops into cardiac dysfunction.

In Vivo Evaluation of Customized Pulse Oximeter and Sensitivity Pulp Tests for Assessment Of Pulp Vitality.

OBJECTIVE: The purpose of this study was to test a customized pulse oximeter (CPO) for evaluation of pulp vitality in primary and permanent teeth against clinical diagnosis (vital and untreated non-vital) in order to expand its clinical use for pulp preservation. STUDY DESIGN: CPO was evaluated on intact primary and permanent central or lateral incisor (CI, LI) teeth-vital (group 1, 20n each); untreated non-vital (group 2, 10n each) and; root filled non-vital (group 3, 10n each) of children 4-12 years according to inclusion/ exclusion criteria. For each patient CPO was first applied on finger followed by vitality tests in following sequence-electrical, pulse oximetry and thermal tests. RESULTS: Mean oxygen saturation (%SpO2) in permanent and primary-vital teeth was 88.78% & 87.77% respectively; non-vital teeth was 74.67% & 75.00% respectively; and in all root filled teeth was 0%. Tooth and finger oxygen saturation values showed strong positive relationship in vital primary or permanent teeth and; no correlation in untreated non-vital primary or permanent teeth. The accuracy rate of thermal pulp test and pulse oximetry was 100% and for electrical pulp test it was 90% for permanent and 86.67% for primary teeth. CONCLUSION: The CPO tested in this study proved to be a valuable adjunct for diagnosing pulp vitality by objective means.

Histologic validation of ICDAS-II and polarization sensitive optical coherence tomography to detect smooth surface early carious lesions.

AIM: This in vitro study aimed to histologically validate and compare the methods for detection of smooth surface early carious lesions (ECLs) that is, International caries detection and assessment system for the smooth surface (ICDAS-II-SSC), Polarization sensitive optical coherence tomography (PS-OCT), and radiography. METHODOLOGY: PS-OCT images for scores 0-3 of ICDAS-II-SSC were standardized according to ECLs' depth. Preliminary PS-OCT images for ICDAS-II-SSC score-2 of pigmented ECLs showed reduced lesion depth and therefore were dichotomized into scores 2 and 2p for white and pigmented lesions (ICDAS-II-SSCm). ECLs on one hundred freshly extracted teeth were scored by three examiners for ICDAS-II-SSCm, PS-OCT, radiography, and histology. RESULTS: Compared to histology, ICDAS-II-SSCm showed a strong positive correlation followed by PS-OCT and radiographic evaluation. ICDAS-II-SSCm had a strong positive correlation with PS-OCT, while both variables had a weak positive correlation with radiography. PS-OCT detected the activity of ECLs by directly relating the image depth of ECLs to their mineral volume content. CONCLUSION: The current scope of ICDAS-II should be reviewed since the pigmentation can be misinterpreted as an active lesion. Till then, ICDAS-II-SSC is an effective visual method for early caries detection. PS-OCT has the potential to become a probe with the proper algorithm for diagnostic purposes.

Infective Larvae of Brugia malayi Induce Polarization of Host Macrophages that Helps in Immune Evasion.

Filarial parasites suppress, divert, or polarize the host immune response to aid their survival. However, mechanisms that govern the polarization of host MΦs during early filarial infection are not completely understood. In this study, we infected BALB/c mice with infective larvae stage-3 of Brugia malayi (Bm-L3) and studied their effect on the polarization of splenic MΦs. Results showed that MΦs displayed M2-phenotype by day 3 p.i. characterized by upregulated IL-4, but reduced IL-12 and Prostaglandin-D2 secretion. Increased arginase activity, higher arginase-1 but reduced NOS2 expression and poor phagocytic and antigen processing capacity was also observed. M2 MΦs supported T-cell proliferation and characteristically upregulated p-ERK but downregulated NF-κB-p65 and NF-κB-p50/105. Notably, Bm-L3 synergized with host regulatory T-cells (Tregs) and polarized M2 MΦs to regulatory MΦs (Mregs) by day 7 p.i., which secreted copious amounts of IL-10 and prostaglandin-E2. Mregs also showed upregulated expression levels of MHC-II, CD80, and CD86 and exhibited increased antigen-processing capacity but displayed impaired activation of NF-κB-p65 and NF-κB-p50/105. Neutralization of Tregs by anti-GITR + anti-CD25 antibodies checked the polarization of M2 MΦs to Mregs, decreased accumulation of regulatory B cells and inflammatory monocytes, and reduced secretion of IL-10, but enhanced IL-4 production and percentages of eosinophils, which led to Bm-L3 killing. In summary, we report hitherto undocumented effects of early Bm-L3 infection on the polarization of splenic MΦs and show how infective larvae deftly utilize the functional plasticity of host MΦs to establish themselves inside the host.

New Drugs and Vaccines for Tuberculosis.

BACKGROUND: Tuberculosis (TB) is an infectious disease that resulted in estimated 9.6 million new cases in 2014 and 1.5 million deaths. The available drug regimen for TB is time consuming which more often leads to the patient non compliance which then results in occurrence of drug resistant TB (Multi-drug and extremely drug resistant TB) in several portions of the world. METHODS: The dangerous combinations of TB and HIV is taking its toll on human health. The foremost factor is non- profit associated with the development of anti TB drugs. There is almost 10 different drugs in various levels of trials whereas the vaccine development is focusing more on adult vaccine rather than a child vaccine. RESULTS: More than 15 vaccine candidate are in various stages of pipelines. Present compilation gives an account for various drug candidates and vaccine products in various stages of drug development. Also included is a recent collection of patents for assay methods, potential drug candidates/classes and vaccination products. CONCLUSION: The need is for improvement in the activity and chemical and biological description of under development compounds. Lastly the set up for clinical and appropriate uses for running a reliable clinical trial is a necessary prerequisite.

Discovery of novel inhibitors for Leishmania nucleoside diphosphatase kinase (NDK) based on its structural and functional characterization.

Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100 mM Bis-Tris propane pH 6.0 and 50 mM MgCl2. It belongs to the hexagonal space group P6322 with unit cell parameters a = b = 115.18, c = 62.18 Å and α = ß = 90°, γ = 120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and Rfree values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation-based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed ~45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.

Identification of Novel Inhibitors of Leishmania donovani γ-Glutamylcysteine Synthetase Using Structure-Based Virtual Screening, Docking, Molecular Dynamics Simulation, and in Vitro Studies.

Trypansomatids maintain their redox balance by the trypanothione-based redox system, enzymes of which exhibit differences from mammalian homologues. γ-Glutamylcysteine synthetase (Gcs) is an essential enzyme in this pathway that performs the first and rate-limiting step. l-Buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of Gcs, induces toxicity in hosts infected with Trypanosoma brucei, underlining the need for novel Gcs inhibitors. The present study reports identification of Leishmania donovani Gcs (LdGcs) inhibitors using computational approaches and their experimental validation. Analysis of inhibitor-LdGcs complexes shows modifications that could result in increased efficacy of these compounds.

CaMKIIδ subtypes differentially regulate infarct formation following ex vivo myocardial ischemia/reperfusion through NF-κB and TNF-α.

Deletion of Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) has been shown to protect against in vivo ischemia/reperfusion (I/R) injury. It remains unclear which CaMKIIδ isoforms and downstream mechanisms are responsible for the salutary effects of CaMKIIδ gene deletion. In this study we sought to compare the roles of the CaMKIIδB and CaMKIIδC subtypes and the mechanisms by which they contribute to ex vivo I/R damage. WT, CaMKIIδKO, and mice expressing only CaMKIIδB or δC were subjected to ex vivo global ischemia for 25min followed by reperfusion. Infarct formation was assessed at 60min reperfusion by triphenyl tetrazolium chloride (TTC) staining. Deletion of CaMKIIδ conferred significant protection from ex vivo I/R. Re-expression of CaMKIIδC in the CaMKIIδKO background reversed this effect and exacerbated myocardial damage and dysfunction following I/R, while re-expression of CaMKIIδB was protective. Selective activation of CaMKIIδC in response to I/R was evident in a subcellular fraction enriched for cytosolic/membrane proteins. Further studies demonstrated differential regulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and tumor necrosis factor alpha (TNF-α) expression by CaMKIIδB and CaMKIIδC. Selective activation of CaMKIIδC was also observed and associated with NF-κB activation in neonatal rat ventricular myocytes (NRVMs) subjected to oxidative stress. Pharmacological inhibition of NF-κB or TNF-α significantly ameliorated infarct formation in WT mice and those that re-express CaMKIIδC, demonstrating distinct roles for CaMKIIδ subtypes in I/R and implicating acute activation of CaMKIIδC and NF-κB in the pathogenesis of reperfusion injury.

Biotransformation of lignan glycoside to its aglycone by Woodfordia fruticosa flowers: quantification of compounds using a validated HPTLC method.

CONTEXT: Saraca asoca Linn. (Caesalpiniaceae) is an important traditional remedy for gynaecological disorders and it contains lyoniside, an aryl tetralin lignan glycoside. The aglycone of lyoniside, lyoniresinol possesses structural similarity to enterolignan precursors which are established phytoestrogens. OBJECTIVES: This work illustrates biotransformation of lyoniside to lyoniresinol using Woodfordia fruticosa Kurz. (Lythraceae) flowers and simultaneous quantification of lyoniside and lyoniresinol using a validated HPTLC method. MATERIALS AND METHODS: The aqueous extract prepared from S. asoca bark was fermented using W. fruticosa flowers. The substrate and fermented product both were simultaneously analyzed using solvent system:toluene:ethyl acetate:formic acid (4:3:0.4) at 254 nm. The method was validated for specificity, accuracy, precision, linearity, sensitivity and robustness as per ICH guidelines. RESULTS: The substrate showed the presence of lyoniside, however, it decreased as the fermentation proceeded. On 3rd day, lyoniresinol starts appearing in the medium. In 8 days duration most of the lyoniside converted to lyoniresinol. The developed method was specific for lyoniside and lyoniresinol. Lyoniside and lyoniresinol showed linearity in the range of 250-3000 and 500-2500 ng. The method was accurate as resulted in 99.84% and 99.83% recovery, respectively, for lyoniside and lyoniresinol. CONCLUSION: Aryl tetralin lignan glycoside, lyoniside was successfully transformed into lyoniresinol using W. fruticosa flowers and their contents were simultaneously analyzed using developed validated HPTLC method.

Development of Leishmania donovani stably expressing DsRed for flow cytometry-based drug screening using chalcone thiazolyl-hydrazone as a new antileishmanial target.

Green fluorescent protein produces significant fluorescence and is extremely stable, however its excitation maximum is close to the ultraviolet range and thus can damage living cells. Hence, Leishmania donovani stably expressing DsRed were developed and their suitability for flow cytometry-based antileishmanial screening was assessed by evaluating the efficacies of standard drugs as well as newly synthesised chalcone thiazolyl-hydrazone compounds. The DsRed gene was successfully integrated at the 18S rRNA locus of L. donovani and transfectants (LdDsRed) were selected using hygromycin B. Enhanced expression of DsRed and a high level of infectivity to J774A.1 macrophages were achieved, which was confirmed by fluorescence microscopy and flow cytometry. Furthermore, these LdDsRed transfectants were utilised for development of an in vitro screening assay using the standard antileishmanial drugs miltefosine, amphotericin B, pentamidine and paromomycin. The response of transfectants to standard drugs correlated well with previous reports. Subsequently, the suitability of this system was further assessed by screening a series of 18 newly synthesised chalcone thiazolyl-hydrazone compounds in vitro for their antileishmanial activity, wherein 8 compounds showed moderate antileishmanial activity. The most active compound 5g, with ca. 73% splenic parasite reduction, exerted its activity via generating nitric oxide and reactive oxygen species and inducing apoptosis in LdDsRed-infected macrophages. Thus, these observations established the applicability of LdDsRed transfectants for flow cytometry-based antileishmanial screening. Further efforts aimed at establishing a high-throughput screening assay and determining the in vivo screening of potential antileishmanial leads are required.