Leptospirosis in central & eastern Uttar Pradesh, an underreported disease: A prospective cross-sectional study.

Background & objective: Leptospirosis is a zoonotic disease associated with potentially fatal consequences and a grossly underreported disease in Uttar Pradesh. However, only a few studies are available which report the prevalence of leptospirosis in this State. Hence, this study was undertaken to know the status of the disease in central and eastern Uttar Pradesh. Methods: A total of 143 serum and urine samples were collected from patients with acute febrile illness from July 2017 to March 2019. All the serum samples were tested for Leptospira by rapid IgM antibody card and IgM ELISA and urine samples were tested by real-time polymerase chain reaction (RT-PCR) to detect Leptospira DNA. All positive and 10 per cent negative sera from ELISA and RT-PCR (all rapid test positive were also ELISA positive) were sent to the ICMR-Regional Medical Research Centre, Port Blair for microscopic agglutination test (MAT). Results: Thirty eight (26.6%) out of 143 samples were positive for leptospirosis either by ELISA or RT-PCR. Positive results were eight (6%) by Rapid card, 32 (22%) by IgM ELISA, 10 (7%) by MAT, 10 (7%) by RT-PCR. In MAT, the most common serovar was Lai followed by Hebdomadis, Bangkinang and Pomona. Interpretation & conclusions: Leptospirosis was found to be one of the important causes for acute febrile illness in the central and eastern parts of Uttar Pradesh. The results of the present study suggest that it is necessary to increase diagnostic facility and awareness in clinicians for the screening of leptospirosis in acutely febrile patients to decrease morbidity and mortality associated with this disease.

Liposome based drug delivery as a potential treatment option for Alzheimer's disease.

Alzheimer's disease is a neurodegenerative condition leading to atrophy of the brain and robbing nearly 5.8 million individuals in the United States age 65 and older of their cognitive functions. Alzheimer's disease is associated with dementia and a progressive decline in memory, thinking, and social skills, eventually leading to a point that the individual can no longer perform daily activities independently. Currently available drugs on the market temporarily alleviate the symptoms, however, they are not successful in slowing down the progression of Alzheimer's disease. Treatment and cures have been constricted due to the difficulty of drug delivery to the blood-brain barrier. Several studies have led to identification of vesicles to transport the necessary drugs through the blood-brain barrier that would typically not achieve the targeted area through systemic delivered medications. Recently, liposomes have emerged as a viable drug delivery agent to transport drugs that are not able to cross the blood-brain barrier. Liposomes are being used as a component of nanoparticle drug delivery; due to their biocompatible nature; and possessing the capability to carry both lipophilic and hydrophilic therapeutic agents across the blood brain barrier into the brain cells. Studies indicate the importance of liposomal based drug delivery in treatment of neurodegenerative disorders. The idea is to encapsulate the drugs inside the properly engineered liposome to generate a response of treatment. Liposomes are engineered to target specific diseased moieties and also several surface modifications of liposomes are under research to create a clinical path to the management of Alzheimer's disease. This review deals with Alzheimer's disease and emphasize on challenges associated with drug delivery to the brain, and how liposomal drug delivery can play an important role as a drug delivery method for the treatment of Alzheimer's disease. This review also sheds some light on variation of liposomes. Additionally, it emphasizes on the liposomal formulations which are currently researched or used for treatment of Alzheimer's disease and also discusses the future prospect of liposomal based drug delivery in Alzheimer's disease.

Evaluating the efficiency of specimen (sample) pooling for real-time PCR based diagnosis of COVID-19.

PURPOSE: This study is aims at evaluating the efficacy and sensitivity of specimen pooling for testing of SARS-CoV-2 virus to determine the accuracy, resource savings, and identification of borderline positive cases without impacting the accuracy of the testing. METHOD: This study was conducted between August and October 2020, we performed COVID-19 testing by RT-PCR on the samples from varying prevalence of rural population (non-hot spot) referred to COVID laboratory, in the first step, the samples were collated into pools of 5 or 10. These pools were tested by RT-PCR. Negative pools were reported as negative whereas positive pools of 5 and 10 were then de-convoluted and each sample was tested individually. RESULTS: In the present study, we tested 1580 samples in 158 pools of 10 and 17,515 samples in 3503 pools of 5. Among 10 samples pool, 11 (13%) pools flagged positive in the first step. In the second step, among 11 pools (110 samples) de-convoluted strategy was followed in which 10 individual samples came positive. Among 5 samples pool, 164 (13%) pools flagged positive in the first step. In the second step, among 164 pools (820 samples) de-convoluted strategy was followed in which 171 individual samples came positive. The pooled sample testing strategy saves substantial resources and time during surge testing and enhanced pandemic surveillance. This approach requires around 76%-93% fewer tests in low to moderate prevalence settings and group sizes up to 5-10 in a population, compared to individual testing. CONCLUSION: Pooled sample RT- PCR analysis strategies can save substantial resources and time for COVID-19 mass testing in comparison with individual testing without compromising the quality of outcome of the test. In particular, the pooled sample approach can facilitate mass screening in the early asymptomatic stages of COVID-19 infections.

Evaluation of TaqMan based real-time PCR assay targeting LipL32 gene for leptospirosis in serologically positive human urine samples from north India.

PURPOSE: Rapid diagnosis of leptospirosis, through culture and/or serology, can be difficult without proper expertise and is often delayed because of the length of time required to obtain results. In India, especially in north India; leptospirosis is a grossly underreported disease, probably due to the lack of diagnostic modalities and lack of awareness of the disease among physicians. In this study, we aimed to identify leptospires in urine samples from serologically positive patients of leptospirosis through TaqMan based Real-time PCR using the LipL32 gene. Although environmental conditions are suitable in north India nowadays, very few studies were there and none by real-time PCR. METHOD: A total of 50 urine and blood samples (40 seropositive and 10 seronegative by IgM ELISA) were recruited in the study. Urine samples were tested by real-time PCR to detect Leptospira DNA and blood samples were used for microscopic agglutination test (MAT) evaluation. RESULT: We were able to detect leptospires in 20% (10/50) cases using our real-time PCR assay. In all 10 PCR positive urine samples, the LipL32 gene was detected and the lower limit of detection in L. interrogans DNA was found to be 20 GE/µL with a 95% cutoff value. CONCLUSION: We conclude that this real-time PCR assay with high specificity and sensitivity less prone to contamination may prove to be a promising approach for diagnosing acute leptospires in urine samples in the second week of acute febrile illness due to leptospiruria starts approximately in the second week of illness.

Scrub typhus: An under-reported and emerging threat - hospital based study from central and eastern Uttar Pradesh, India.

BACKGROUND & OBJECTIVES: Scrub typhus is a zoonotic rickettsial disease that is transmitted by the bite of the larval stage (chiggers) of trombiculid mites. The aim of this study was to determine the existence of scrub typhus in central and eastern Uttar Pradesh, India in patients with acute febrile illness (AFI) presenting to a super specialty tertiary level institute. METHODS: This prospective hospital-based study was conducted for a period of one year, from August 2018 to July 2019. About 2-5 mL of blood samples, along with clinical, epidemiological, and demographic data from a total of 125 patients presenting with acute febrile illness to outpatient and inpatient departments, were collected. ELISA testing tested the sera from blood samples for IgM antibodies against scrub typhus. Samples were also tested for dengue, leptospirosis, malaria and typhoid. RESULTS: During the study period, out of a total of 125 samples collected, 20% were found positive for IgM antibodies against scrub typhus. Demographically higher positivity was found in males, older age group, and in rural area. Rainfall was found to be important epidemiological parameter for presence of scrub typhus. Co-infection with dengue, leptospirosis and malaria was found. INTERPRETATION & CONCLUSION: Scrub typhus is found to be an important cause of acute febrile illness. It is necessary to include it in differential diagnosis of AFI cases even in absence of eschar. Diagnostic facilities of this as a screening test should be started in primary care centers or community health centers of rural areas of districts of central and eastern Uttar Pradesh, India.

Histone Deacetylases Inhibitors in Neurodegenerative Diseases, Neuroprotection and Neuronal Differentiation.

Histone deacetylases (HADC) are the enzymes that remove acetyl group from lysine residue of histones and non-histone proteins and regulate the process of transcription by binding to transcription factors and regulating fundamental cellular process such as cellular proliferation, differentiation and development. In neurodegenerative diseases, the histone acetylation homeostasis is greatly impaired, shifting towards a state of hypoacetylation. The histone hyperacetylation produced by direct inhibition of HDACs leads to neuroprotective actions. This review attempts to elaborate on role of small molecule inhibitors of HDACs on neuronal differentiation and throws light on the potential of HDAC inhibitors as therapeutic agents for treatment of neurodegenerative diseases. The role of HDACs in neuronal cellular and disease models and their modulation with HDAC inhibitors are also discussed. Significance of these HDAC inhibitors has been reviewed on the process of neuronal differentiation, neurite outgrowth and neuroprotection regarding their potential therapeutic application for treatment of neurodegenerative diseases.

Role of Edaravone as a Treatment Option for Patients with Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig's disease, is a progressive and fatal neurodegenerative disease that leads to a loss of muscle control due to nerve cells being affected in the brain and spinal cord. Some of the common clinical presentations of ALS include weakness of muscles, changes in behavior, dysfunction in speech, and cognitive difficulties. The cause of ALS is uncertain, but through several studies, it is known that mutations in SOD1 or C9orf72 genes could play a role as a factor of ALS. In addition, studies indicate that an excessive amount of free radicals, the reactive oxygen species (ROS), leads to neuronal damage by the peroxidation of unsaturated fatty acids in the neuronal cells. Edaravone, the newly approved antioxidant drug for ALS, halts the progression of ALS in the early stages through its cytoprotective effect and protects the nerves by reducing ROS. In this review, different aspects of ALS will be discussed, including its pathology, genetic aspect, and diagnosis. This review also focuses on edaravone as a treatment option for ALS, its mechanism of action, and its pharmacological properties. Clinical trials and adverse effects of edaravone and care for ALS patient are also discussed.

Retina Compatible Interactions and Effective Modulation of Blood Ocular Barrier P-gp Activity by Third-Generation Inhibitors Improve the Ocular Penetration of Loperamide.

Effective drug delivery to the deeper ocular tissues remains an unresolved conundrum mainly due to the expression of multidrug resistance efflux proteins, besides tight junction proteins, in the blood ocular barriers (BOBs). Hence, the purpose of the current research was to investigate the ability of the third-generation efflux protein inhibitors, elacridar (EQ), and tariquidar (TQ), to diminish P-glycoprotein (P-gp) mediated efflux transport of loperamide (LOP), a P-gp substrate, across the BOB in Sprague Dawley rats. Initially, Western blot analysis confirmed the expression of P-gp in the iris-ciliary bodies and the retina choroid in the wild type rats. Next, the ocular distribution of LOP, in the presence and absence of EQ/TQ (at 2 doses), was evaluated. The significantly higher aqueous humor/plasma (DAH) and vitreous humor (VH)/plasma (DVH) distribution ratios of LOP in the rats pretreated with EQ or TQ demonstrated effective inhibition of P-gp activity in the BOB. Interestingly, the modulation of P-gp activity by EQ/TQ was more pronounced at the lower dose. The normal functioning and architecture of the retina, as indicated by electroretinography studies, confirmed the cytocompatibility of LOP and EQ/TQ interactions at the doses tested.

A Combined In Vitro Assay for Evaluation of Neurotrophic Activity and Cytotoxicity.

Neurotrophic assays are phenotypic methods to identify molecules that stimulate differentiation of neuronal cells. Bioactive small molecules with neurotrophic actions hold great promise as therapeutic agents for the treatment of neurodegenerative diseases and neuronal injuries by virtue of their ability to stimulate neuritic outgrowth. A combined in vitro method, which measures neurotrophic activity and cytotoxicity in a single assay, has been described. This assay, performed in 96-well microplates with PC12 and Neuroscreen-1 (NS-1; a subclone of PC12) cells, is a simple tool for identification of new neurotrophic agents. Stimulation of neurite outgrowth was measured with NIS software by analysis of digital cell images as multiple parameters, namely, mean neurite length, neurite length/cell, nodes/cell, and number of neurites/cell. The assay has been standardized and validated with dose-response analysis for nerve growth factor (NGF) and mechanism-based inhibitors of NGF-induced neurite outgrowth, namely, SU6656 (an Src family kinase inhibitor) and PD98059 (a MEK inhibitor). The assay has been successfully applied for screening natural and synthetic compound libraries for cytotoxicity and neurotrophic activity. Screening of a set of harmala alkaloids identified harmine as a potential neurotrophic molecule that significantly stimulated NGF-induced neurite outgrowth in the NS-1 cells. Important advantages of this method are its simplicity and determination of cytotoxicity and neurotrophic activity in a single assay. This assay may be suitable for primary and cultured neuronal cells.

Mechanism for neurotropic action of vorinostat, a pan histone deacetylase inhibitor.

In this study we investigated the neurotrophic actions of vorinostat (suberoylanilide hydroxamic acid, SAHA), a class I and class II HDAC inhibitor, on the differentiation of Neuroscreen-1 (NS-1) cells. NS-1 cell is a subclone of the rat pheochromocytoma cell line (PC 12). Vorinostat independently induced neurite outgrowth in NS-1 cells. The NS-1 cells were further interrogated for the effects of vorinostat on intracellular neurotrophin signaling pathways, to understand its mechanism of neurotrophic action. Selective inhibitors of MEK1/2 (PD98059 and U0126), phosphoinositide 3-kinase (PI3K) (LY294002) and tyrosine kinase A (TrkA) (GW441756) were employed for these interrogations. Our results suggest that neurite outgrowth mediated by both nerve growth factor (NGF), an intrinsic neurotrophin, and vorinostat were blocked by the inhibitors of MEK1/2 & PI3K. Vorinostat induced phosphorylation of ERK1/2 occurs at 2h post treatment. Phosphorylation of ERK was abolished in presence of U0126, further confirming the role of ERK pathway in vorinostat-induced differentiation of NS-1 cells. Vorinostat-induced neurite outgrowth also involves the activation of upstream extracellular kinase TrkA, as both vorinostat mediated neurite outgrowth and activation of ERK were attenuated in presence of the TrkA inhibitor, GW441756. Vorinostat also stimulated hyperacetylation of α-tubulin and histones H3/H4 in NS-1 cells. The results suggest that vorinostat exerts a positive effect on the neuritogenesis via activation of MEK1/2 & PI3K pathways involving an upstream kinase, TrkA. Bioactive small molecules with neurotrophic and neuritogenic actions, like vorinostat identified in the present study, hold great promise as therapeutic agents for treatment of neurodegenerative diseases and neuronal injuries by virtue of their ability to stimulate neuritic outgrowth.

Comparison of specimen adequacy and smear quality in oral smears prepared by manual liquid-based cytology and conventional methods.

BACKGROUND: Liquid-based cytology (LBC), recommended in the mass screening of potentially malignant cervical and oral lesions, suffers from high cost owing to the use of expensive automated devices and materials. Considering the need for cost-effective LBC techniques, we evaluated the efficacy of an inexpensive manual LBC (MLBC) technique against conventional cytological technique in terms of specimen adequacy and smear quality of oral smears. MATERIALS AND METHODS: Cytological samples were collected from 21 patients using a cytobrush device. After preparation of a conventional smear, the brush containing the remaining sample was immersed in the preservative vial. The preserved material was processed by an MLBC technique and subsequently, direct smears were made from the prepared cell button. Both conventional and MLBC smears were stained by routine Papanicolaou technique and evaluated by an independent observer for the thickness of the smear, cellular distribution, resolution/clarity of cells, cellular staining characteristics and the presence of unsatisfactory background/artifacts. Each parameter was graded as satisfactory; or satisfactory, but limited; or unsatisfactory. Chi-square test was used to compare the values obtained (significance set at P ≤ 0.05). RESULTS: MLBC technique produced a significant number of satisfactory smears with regard to cell distribution, clarity/resolution, staining characteristics and background/artifacts compared to conventional methods. CONCLUSIONS: MLBC is a cost-effective cytological technique that may produce oral smears with excellent cytomorphology and longer storage life.