Caveolin-1 knockout mitigates breast cancer metastasis to the lungs via integrin α3 dysregulation in 4T1-induced syngeneic breast cancer model.

Caveolin-1 (Cav-1) is a critical lipid raft protein playing dual roles as both a tumor suppressor and promoter. While its role in tumorigenesis, progression, and metastasis has been recognized, the explicit contribution of Cav-1 to the onset of lung metastasis from primary breast malignancies remains unclear. Here, we present the first evidence that Cav-1 knockout in mammary epithelial cells significantly reduces lung metastasis in syngeneic breast cancer mouse models. In vitro, Cav-1 knockout in 4T1 cells suppressed extracellular vesicle secretion, cellular motility, and MMP secretion compared to controls. Complementing this, in vivo analyses demonstrated a marked reduction in lung metastatic foci in mice injected with Cav-1 knockout 4T1 cells as compared to wild-type cells, which was further corroborated by mRNA profiling of the primary tumor. We identified 21 epithelial cell migration genes exhibiting varied expression in tumors derived from Cav-1 knockout and wild-type 4T1 cells. Correlation analysis and immunoblotting further revealed that Cav-1 might regulate metastasis via integrin α3 (ITGα3). In silico protein docking predicted an interaction between Cav-1 and ITGα3, which was confirmed by co-immunoprecipitation. Furthermore, Cav-1 and ITGα3 knockdown corroborated its role in metastasis in the cell migration assay.

Sexual dimorphism in neurobehavioural phenotype and gut microbial composition upon long-term exposure to structural analogues of bisphenol-A.

Bisphenol S (BPS) and Bisphenol F (BPF), the analogues of the legacy endocrine disrupting chemical, Bisphenol A (BPA) are ubiquitous in the environment and present in various consumer goods, and potentially neurotoxic. Here, we studied sex-specific responses of bisphenols on behavioural phenotypes, including their association with pro-inflammatory biomarkers and altered neurotransmitters levels, and the key gut microbial abundances. Neurobehavioural changes, using standard test battery, biochemical and molecular estimations for inflammatory cytokines, neurotransmitters, and oxido-nitrosative stress markers, gene expression analysis using qRT-PCR, H&E based histological investigations, gut permeability assays and Oxford Nanopore-based 16S-rRNA metagenomics sequencing for the gut microbial abundance estimations were performed. Bisphenol(s) exposure induces anxiety and depression-like behaviours, particularly in the male mice, with heightened pro-inflammatory cytokines levels and systemic endotoxemia, altered monoamine neurotransmitters levels/turnovers and hippocampal neuronal degeneration and inflammatory responses in the brain. They also increased gut permeability and altered microbial diversity, particularly in males. Present study provides evidence for sex-specific discrepancies in neurobehavioural phenotypes and gut microbiota, which necessitate a nuanced understanding of sex-dependent responses to bisphenols. The study contributes to ongoing discussions on the multifaceted implications of bisphenols exposure and underscores the need for tailored regulatory measures to mitigate potential health risks associated with them.

Metagenomics analysis of mice gut microbiome to unravel the role of metal exposure and piperine.

The gut and intestinal microbiota consists of trillions of microorganisms inhabiting the human gastrointestinal tract. It plays a crucial role in human health leading to understanding the dynamic crosstalk of host-microbe interaction in the gut and has become necessary for the detection, prevention, or therapy of diseases. Gut microbiota deviations are linked with many diseases, suggesting that various pathways involved in immunity, energy, lipid, and glucose metabolism are affected. Further, it is also altered by external insults such as metal toxicity, antibiotics and pesticides. Heavy metals like arsenic, mercury, cadmium and chromium are some of the well-studied classes of environmental pollutants. Mouse models have become the model of choice for most studies in this emerging field, as they allow perturbations in the gut microbiota to be studied in a controlled experimental setup. Here, we investigate the composition and diversity of intestinal microbes utilizing cecal samples from different intervention groups: arsenic exposure (As(III)), arsenic and piperine co-administration (As +Pp), piperine per se and control group. We obtained DNA samples from these groups and performed PCR amplification and sequencing of the 16S V3-V4 region. The findings showed shift in microbial composition and abundance among different intervention groups, revealing taxa that may contribute to the microbial diversity.

Cidofovir in Severe Hypoxemic Adenoviral Pneumonia.

The authors present a 16-mo-old boy with flu like symptoms, not responding to supportive management and progressed to severe hypoxemic pneumonia. Adenovirus was detected in the nasopharyngeal aspirate. He showed rapid improvement after intravenous cidofovir administration.

Combining donepezil and memantine via mannosylated PLGA nanoparticles for intranasal delivery: Characterization and preclinical studies.

The current work is focused on developing mannose-coated PLGA nanoparticles for delivering Donepezil and Memantine in one dosage form. The formulated nanoparticles were prepared using a simple emulsification technique. The final coated NPs exhibited 179.4 nm size and - 33.1 mV zeta potential and spherical shape. The concentration of IN-administrated MEM and DPZ mannose coated NPs in brain was ~573 and 207 ng/mL respectively. This amount accounts for 3 times more in comparison to uncoated NPs administered via intranasal and peroral routes. The plasma concentration of coated NPs administered via the intranasal route was various times less in comparison to other groups. In the field of pharmacodynamics, the administration of coated NPs via the IN route has shown superior efficacy in comparison to other groups in various investigations involving neurobehavioral assessments, gene expression analyses and biochemical estimations. The findings indicate that the IN route may be a potential avenue for delivering therapeutic agents using nanoparticles to treat neurological illnesses. This approach shows promise as a viable alternative to traditional dose forms and administration methods.

Simultaneous Intranasal Codelivery of Donepezil and Memantine in a Nanocolloidal Carrier: Optimization, Pharmacokinetics, and Pharmacodynamics Studies.

This work focuses on developing nanoemulsions using a low-energy emulsification method for the codelivery of donepezil and memantine in one dosage form intended to be administered via the intranasal route for enhanced brain delivery. The nanoemulsion formulation was prepared using a low emulsification technique and characterized using various microscopy and nasal ciliotoxicity studies. The safe nanoemulsion was intended for preclinical pharmacokinetics with brain distribution and pharmacodynamics in a scopolamine-induced murine model. The formulated nanoemulsion was 16 nm in size, with a zeta potential of -7.22 mV, and exhibited a spherical shape. The brain concentration of IN-administered NE for DPZ and MEM was ∼678 and 249 ng/mL after 15 min. This concentration is more than 2 times higher in amount when compared with NE administered via PO, free drug solution administered via IN and PO route both. However, the plasma concentration of IN-administered NE for DPZ and MEM was ∼3 and 28 ng/mL after 15 min. In pharmacodynamic studies, the efficacy of NE administered via the IN route was higher when compared with other groups in neurobehavioral, biochemical estimation, and gene expression studies. The results suggest that the IN route can be explored in the future for the delivery of actives via nanocolloidal carriers in the brain for neurological disorders and can serve as promising alternatives for conventional dosage forms and routes.

Safety and Outcomes of Midline and Peripherally Inserted Central Catheters in a Pediatric Intensive Care Unit.

OBJECTIVES: We describe our experience with use of midline catheters in PICU and compare the performance of midline catheters to peripherally inserted central catheters (PICC). METHODS: A review of hospital records was done to including all pediatric patients admitted in the pediatric intensive care unit of a tertiary care centre who underwent placement of midline catheters or PICC, over a period of 18 months (July, 2019 to January, 2021). Patient details, indication, type of catheter and number of attempts at insertion, type and number of infusions administered, dwell time and complications were retrieved from the records. Comparison was made between the midline and PICC groups. RESULTS: The median (IQR) age of children was 7 (3-12) years (75.5% males). 161 midline catheters and 104 PICC were inserted with first attempt success rates of 87.6% and 78.8%, respectively. Median cubital vein was used for majority of the insertions (52.8%). Common complications with midline catheters were pain (n=9, 5.6%), blockage (n=8, 5%) and thrombophlebitis (n=6, 3.7%). Median (interquartile range) dwell time in midline group was 7 (5-10) days. The duration of backflow and dwell time were higher in the PICC group compared to midline group (5.5 vs 3 days; P<0.001 and 9 vs 7 days; P<0.001, respectively). CONCLUSION: Retrospective data showed that midline catheters had good utility in PICU, especially in moderately sick children (PRISM score up to 12), and provide a secure intravenous access, which can last for a week.

Transient Receptor Potential (TRP) based polypharmacological combination stimulates energy expending phenotype to reverse HFD-induced obesity in mice.

BACKGROUND & AIM: Obesity is a worldwide epidemic leading to decreased quality of life, higher medical expenses and significant morbidity. Enhancing energy expenditure and substrate utilization in adipose tissues through dietary constituents and polypharmacological approaches is gaining importance for the prevention and therapeutics of obesity. An important factor in this regard is Transient Receptor Potential (TRP) channel modulation and resultant activation of "brite" phenotype. Various dietary TRP channel agonists like capsaicin (TRPV1), cinnamaldehyde (TRPA1), and menthol (TRPM8) have shown anti-obesity effects, individually and in combination. We aimed to determine the therapeutic potential of such combination of sub-effective doses of these agents against diet-induced obesity, and explore the involved cellular processes. KEY FINDINGS: The combination of sub-effective doses of capsaicin, cinnamaldehyde and menthol induced "brite" phenotype in differentiating 3T3-L1 cells and subcutaneous white adipose tissue of HFD-fed obese mice. The intervention prevented adipose tissue hypertrophy and weight gain, enhanced the thermogenic potential, mitochondrial biogenesis and overall activation of brown adipose tissue. These changes observed in vitro as well as in vivo, were linked to increased phosphorylation of kinases, AMPK and ERK. In the liver, the combination treatment enhanced insulin sensitivity, improved gluconeogenic potential and lipolysis, prevented fatty acid accumulation and enhanced glucose utilization. SIGNIFICANCE: We report on the discovery of therapeutic potential of TRP-based dietary triagonist combination against HFD-induced abnormalities in metabolic tissues. Our findings indicate that a common central mechanism may affect multiple peripheral tissues. This study opens up avenues of development of therapeutic functional foods for obesity.

Seed Community Identification Framework for Community Detection over Social Media.

The social media podium offers a communal perspective platform for web marketing, advertisement, political campaign, etc. It structures like-minded end-users over the explicit group as a community. Community structure over social media is the collaborative group of globally spread users having similar interests regarding a communal topic, product or any other axis. In recent years, researchers have widely used clustering techniques of data mining to structure communities over social media. Still, due to a lack of network and implicit communal information, researchers cannot bind mutually robust and modular community structures. The collaborative features of social media are inherent with implicit and explicit end-users. The explicit nature of both active and passive users is easily extracted from the graphical structure of social media. On the other hand, the degree of information inclusion of implicit features depends upon end-users participation. The Implicit features of frequently active users are diversely available, while integrating passive and silent users' implicit features over the community is tedious. This work proposed a social theory based influence maximization (STIM) framework for community detection over social media. It combines user-generated content with profile information, extracts passive social media users through influence maximization, and provides the user space for influencing inactive users. The STIM framework clusters identical nodes over the maximum influencing node axis based on their graphical parameters such as node degree, node similarity, node reachability, modularity, and node density. This framework also provides the structural, relational and mathematical concept for the functional grouping of like-minded people as a community over social media through social theory. Finally, an evaluation has been carried out over six real-time datasets. It analyses that convolution neural network over STIM structure more dense and modular communities via influence maximization. STIM acquired around 93% modularity and 94% Normalized Mutual Information (NMI), resulting in approximately 2.23% and 5.69% improvements in modularity and NMI, respectively, over the best-acquired result of the benchmark approach.

Short-term trivalent arsenic and hexavalent chromium exposures induce gut dysbiosis and transcriptional alteration in adipose tissue of mice.

BACKGROUND: Inorganic arsenic [As(III)] and hexavalent chromium [Cr(VI)] can potentially affect metabolic functions. These heavy metal(s)/metalloids can also affect the gut microbial architecture which affects metabolic health. Here, we assessed the effects of short-term exposure of As(III) and Cr(VI) on key transcription factors in adipose tissues and on selected gut microbial abundances to understand the possible modulatory role of these toxicants on host metabolic health. METHODS AND RESULTS: qRT-PCR based relative bacterial abundance studies in cecal samples, gene expression analysis for gut wall integrity in ileum and colon and adipogenesis, lipolysis, and thermogenic genes in gonadal white and brown adipose tissue (gWAT and BAT), along with tissue oxidative stress parameters have been performed. As(III) and Cr(VI) exposure reduced beneficial Lactobacilli, Bifidobacteria, Akkermansia, Lachenospiraceae, Fecalibacterium, Eubacterium, and clostridium coccoid group while increasing lipopolysaccharides producing Enterobacteriaceae abundances. It also impaired structural features and expression of key tight junction and mucin production genes in ileum and colon (Cld-2, Cld-4, ZO-1, ZO-2, MUC-2 and - 4). In gWAT it inhibited adipogenesis (PPARγ, FASN, SREBP1a), lipolysis (HSL, ACOX-1), and thermogenesis (UCP-1, PGC1a, PRDM-16, PPARa) related genes expression, whereas in BAT, it enhanced adipogenesis and reduced thermogenesis. These exposures also reduces the endogenous antioxidants levels in these tissues and promote pro-inflammatory cytokines genes expression (TLRs, IL-6, MCP-1). The combinatorial exposure appears to have more deleterious effects. CONCLUSION: These effects of As(III) and Cr(VI) may not directly be linked to their known toxicological effects, instead, more intriguing crosstalk with gut microbial ecosystem hold the key.

Tubercular Tenosynovitis Mimicking a Large Ganglion Cyst.

Introduction: Tubercular tenosynovitis of the wrist and hand, although rare, presents a diagnostic dilemma primarily due to its non-specific clinical presentation, insidious course, and the large number of differentials that mimic it. When the diagnosis is late or delayed, significant bone and tendon complications occur. Large progressive swelling around the wrist in TB endemic countries should raise an early suspicion of being of tubercular etiology and should be high on the list of differential diagnoses. Case Report: A 48-year-old female presented with a large progressive swelling on the volar aspect of the left wrist and palm for 7 months, associated with increasing pain, stiffness, limited wrist range of movements, and weakened grip strength. Magnetic resonance imaging (MRI) revealed synovitis and fluid within flexor tendon synovial sheaths. The patient underwent an excision of the mass in toto and the cut section revealed an irregularly thickened wall with rice bodies within. Histopathological examination was indicative of a large ganglion cyst. GeneXpert MTB/RIF assay detected Mycobacterium tuberculosis. Despite histopathology being inconclusive, a diagnosis of TTS was considered due to the patient's clinical presentation, MRI, and operative findings. The patient was started on an antitubercular drug regimen for a 1-year duration. The patient regained a complete range of movements of fingers and wrist and normal grip strength at the 3rd month follow-up. Conclusion: TTS is a challenging diagnosis entity. The diagnostic confirmation was achieved either by histopathology or detection of the organism either by culture or GeneXpert MTB/RIF assay. When the diagnosis is unsupported, drug therapy can be initiated empirically on strong suspicion of tuberculosis, especially in TB endemic areas.

Therapeutic effects of CoenzymeQ10, Biochanin A and Phloretin against arsenic and chromium induced oxidative stress in mouse (Mus musculus) brain.

Arsenic and chromium are the most common environmental toxicants prevailing in nature. Hence, the present study endeavors to investigate the salutary effects of Coenzyme Q10 (CoQ10), Biochanin A (BCA), and Phloretin (PHL) on the combined neurotoxic impact of arsenic and chromium in the Swiss albino mice (Mus musculus). Sodium meta-arsenite (100 ppm) and potassium dichromate (75 ppm) were given orally in conjugation with CoQ10 (10 mg/kg), BCA & PHL (50 mg/kg each) in accordance with body weight per day for the 2 weeks experimental duration. Weight reduction was figured out in the exposed toxic group of arsenic and chromium in contrast with the comparison group (control), and with the selected anti-oxidants treatment, it rose significantly to the basal status (p < 0.05). The concentration of arsenic and chromium was reduced significantly (p < 0.001) amidst all the natural compounds co-medicated groups. Anti-oxidant indicators, viz. lipid peroxidation (LPO) and protein carbonyl content (PCC), were found elevated, with reduction observed in the levels of superoxide dismutase (SOD), reduced glutathione (GSH), glutathione s-transferase (GST), and total thiols (TT) in the arsenic and chromium, co-exposed mice. The alterations in redox homeostasis were well corroborated with the estimations of cholinesterase's enzymes (p < 0.05) along with DNA fragmentation assay and altered Nrf2 signaling. The administration of CoQ10, BCA, and PHL ameliorated the effects of arsenic and chromium induced oxidative stress in the exposed mice. Our research unfolds the remedial outcome of these natural compounds contrary to the combined arsenic and chromium associated-neurotoxicity in the experimental model.

Recent trends of natural based therapeutics for mitochondria targeting in Alzheimer's disease.

Alzheimer's disease (AD) is the most ubiquitous neurodegenerative disorder with impaired cognitive functionality. Till date, the specific pathophysiology related to AD is still elusive. Recent reports suggest mitochondrial dysfunctionality like oxidative stress, Ca2+ disbalance, apoptosis, decrease energy and its metabolism plays an important. Recent reports about mitochondrial mechanisms and dynamics in AD unravelled new insights of molecular targets. Targeting multi-pathway via natural products may attenuate and prevent the mitochondria dysfunctionality. In this review, we have focused on the pathophysiology events of mitochondrial dysfunction in AD as well as mitochondrial fusion and fission mechanisms, role of aging in multicellular organisms, and how these connect to cell cycle regulation, and transmission of energy status.

Point Prevalence of Peripheral Neuropathy in Children and Adolescents with Type 1 Diabetes Mellitus.

OBJECTIVE: To assess the point prevalence of peripheral neuropathy (PN) in children with type 1 diabetes mellitus (T1DM) and to determine their predictors. METHODS: In this cross-sectional study, children aged 8-18 y with T1DM on insulin therapy for > 2 y and free from acute complications were enrolled. All participants were evaluated for symptoms of PN with diabetic neuropathy symptom (DNS) score and underwent a detailed neurological examination. Assessment of nerve dysfunction was done using nerve conduction studies (NCS). The disease-related factors that increase the risk of PN were determined. RESULTS: Fifty children (52% boys) were enrolled with mean age of 12.2 ± 2.8 y and duration of diabetes 5.1 ± 2.1 y. No subject had clinical evidence or DNS score suggestive of PN. Twenty-eight (56%) children demonstrated subclinical neuropathy on NCS. Proportion of children with pure motor, pure sensory, and mixed motor-sensory neuropathy was 40%, 2%, and 14%, respectively. The peroneal nerve was the most common motor nerve affected. Poor glycemic control (HbA1c > 9%) and longer duration of diabetes (> 5 y) were significantly associated with the risk of PN (p value < 0.05). CONCLUSION: A large proportion of children with T1DM have subclinical PN. Poor glycemic control and longer duration of diabetes are risk factors for nerve dysfunction. Neurophysiological studies should be performed in these children to facilitate early detection.

Allicin, a dietary trpa1 agonist, prevents high fat diet-induced dysregulation of gut hormones and associated complications.

Scope. Given the global epidemic of diabesity (co-existence of both diabetes and obesity), novel approaches that target gut hormone secretion and their modulation may offer the dual benefits of increased efficacy and limited side effects. In the present study, we tested the hypothesis that agonism of Transient Receptor Potential Ankyrin 1 (TRPA1), using a dietary activator, has a modulatory role in high fat diet (HFD)-induced dysregulation of post-prandial gut hormone responses and prevention of metabolic alterations. Methods and results. The effect of HFD on TRPA1 expression in different parts of the gut using immunohistochemistry, western blotting and RT-PCR was studied. Dietary TRPA1 agonist, Allicin Rich Garlic Juice (ARGJ), was co-administered along with HFD in mice for three months and various metabolic health parameters, relative gut hormone levels and inflammation were observed. The HFD caused substantial reduction in gut TRPA1 expression along with dysregulation in post-prandial normalization of gut hormone levels, particularly GLP-1, precipitating hunger phenotype, altered glucose homeostasis, hepatic inflammation and fat accumulation. TRPA1 agonism through ARGJ co-supplementation prevented HFD-induced dysregulation in post-prandial normalization of gut hormone levels and averted metabolic and inflammatory complications in peripheral tissues. Conclusion. Our findings provide evidence that ARGJ (diet-based TRPA1 agonism) can be employed as a feasible strategy, as nutraceuticals or food, to prevent HFD-induced metabolic complications.

Designing, fabrication and evaluation of a rapid, point-of-care and noninvasive system for the detection of lead (Pb2+).

Non-invasive collection of biological sample such as sweat, urine, saliva, hairs and, stool and onsite detection of anlaytes in those samples is an interesting and viable approach for rapid screening of various toxicants in body. Environmental exposure/presence of lead (82Pb) and its rapid detection provide one such opportunity. A chemical spot based colorimetric method and a transdermal patch device based on this spot test, is developed for rapid and qualitative assessment of inorganic lead (Pb2+) in non-coloured biological or environmental liquid samples. The transdermal patch system contains two important parts, a chemical spot prepared on a thin glass sheet and, an absorbent paper (11 µm pore size). A one step colour development reaction is able to identify the presence or absence of Pb2+. In-vitro evaluation for sensitivity and cut-off value determination, within run and between run precision testing, specificity testing were done. In-vivo evaluation of the developed patch system was performed in occupationally lead-exposed subjects and in control volunteers. In-vivo field testing results were further validated with gold standard test for lead detection. Blood lead levels and patch lead levels were found to be positively correlated (r = 0.57, P < 0.0001). In addition, the sensitivity and specificity of device in identification of Pb2+ was found to be 75.93% (95% CI = 62.36%-86.51%) and 95.24% (95% CI = 76.18%-99.88%). The developed system appears as a reliable, non-invasive rapid test with minimum step involve for identification of Pb2+ in a given system.

E-cig vapor condensate alters proteome and lipid profiles of membrane rafts: impact on inflammatory responses in A549 cells.

Electronic cigarettes (e-cigs) are battery-operated heating devices that aerosolize e-liquid, typically containing nicotine and several other chemicals, which is then inhaled by a user. Over the past decade, e-cigs have gained immense popularity among both smokers and non-smokers. One reason for this is that they are advertised as a safe alternative to conventional cigarettes. However, the recent reports of e-cig use associated lung injury have ignited a considerable debate about the relative harm and benefits of e-cigs. The number of reports about e-cig-induced inflammation and pulmonary health is increasing as researchers seek to better understand the effects of vaping on human health. In line with this, we investigated the molecular events responsible for the e-cig vapor condensate (ECVC)-mediated inflammation in human lung adenocarcinoma type II epithelial cells (A549). In an attempt to limit the variables caused by longer ingredient lists of flavored e-cigs, tobacco-flavored ECVC (TF-ECVC±nicotine) was employed for this study. Interestingly, we observed significant upregulation of cytokines and chemokines (IL-6, IL-8, and MCP-1) in A549 cells following a 48 h TF-ECVC challenge. Furthermore, there was a significant increase in the expression of pattern recognition receptors TLR-4 and NOD-1, lipid raft-associated protein caveolin-1, and transcription factor NF-кB in TF-ECVC with and/or without nicotine-challenged lung epithelial cells. Our results further demonstrate the harboring of TLR-4 and NOD-1 in the caveolae of TF-ECVC-challenged A549 cells. Proteomic and lipidomic analyses of lipid raft fractions from control and challenged cells revealed a distinct protein and lipid profile in TF-ECVC (w/wo nicotine)-exposed A549 cells. Interestingly, the inflammatory effects of TF-ECVC (w/wo nicotine) were inhibited following the caveolin-1 knockdown, thus demonstrating a critical role of caveolae raft-mediated signaling in eliciting inflammatory responses upon TF-ECVC challenge. Graphical Abstract Graphical Abstract.

Predictors of severe dengue amongst children as per the revised WHO classification.

BACKGROUND & OBJECTIVES: World Health Organization (WHO) revised its guidelines for classification and management of dengue in 2009. This revised system was found out to have good sensitivity and negative predictive value but poor specificity as well as positive predictive value. METHODS: This retrospective study was carried out in a tertiary care hospital of Delhi, India to assess factors predicting the occurrence of severe dengue in children as per the revised classification. A total of 647 suspected dengue cases were admitted in the hospital in the year 2015. Detailed clinical and epidemiological data of 170 patients who were confirmed as dengue either by NS1 antigen test or by serology (Ig M positive) were recorded and statistically analyzed. RESULTS: The number of laboratory-confirmed cases was 170 and included thirty (17.65%) dengue fever (DF), 106 (62.35%) dengue with warning signs (DWS) and 34 (20.0%) severe dengue (SD) patients. Regression analysis revealed that presence of vomiting, altered sensorium, shock, peri-orbital edema, hepatomegaly, splenomegaly, severe anemia, thrombocytopenia, elevated urea and creatinine, decreased total protein and globulin were significantly associated with occurrence of severe disease. INTERPRETATION & CONCLUSION: The addition of clinical features (peri-orbital edema and splenomegaly); and laboratory findings (elevated urea and creatinine, decreased serum protein and globulin) might help improve the sensitivity and specificity of the revised WHO dengue classification in predicting severe dengue.