Multi-element Contamination and Health Risks in Green Leafy Vegetables from Ambagarh Chowki, Chhattisgarh, India.

Leafy plants are commonly consumed as vegetables in India due to their high nutrient and vitamin content. This study, conducted in Ambagarh Chowki (India), investigated the accumulation potential of 52 elements (including Al, As, Ba, Be, Bi, Ca, Cd, Ce, Co, Cr, Cu, Dy, Er, Eu, Fe, Ga, Gd, Ge, Ho, K, La, Li, Lu, Mg, Mn, Mo, Na, Nb, Nd, Ni, P, Pb, Pr, Rb, Sb, Sc, Se, Sm, Sn, Sr, Tb, Te, Th, Ti, Tl, Tm, U, V, W, Y, Yb, and Zn) in seven leafy vegetable species, namely Amaranthus tricolor L., Corchorus olitorius L., Cordia myxa L., Hibiscus sabdariffa L., Ipomoea batatas (L.) Lam., Moringa oleifera Lam., and Spinacia oleracea L. Technique: Inductively coupled plasma mass spectrometry (ICP-MS) was employed for analysis. The maximum concentrations of elements such as Al, Ba, Be, Bi, Cd, Co, Cr, Fe, Ga, Ge, Li, Mn, Ni, Pb, Sb, Th, Tl, U, V, W, and REEs were observed in S. oleracea leaves, indicating their highest accumulation potential. In contrast, the maximum concentrations of As were found in H. sabdariffa leaves; Ca and Si in M. oleifera leaves; Mg, Sr, and Mo in A. tricolor leaves; and P, K, Cu, and Zn in C. myxa leaves, respectively. Twenty-one elements (Cr, Cd, Pb, Ni, Co, V, Cu, Zn, Fe, Mn, Th, Sb, Ba, Be, Li, Sr, Tl, U, Se, Sn, and REEs) exceeded permissible limits set by the WHO. The elevated hazard index values indicated significant non-carcinogenic effects. The sources of these elements could be attributed to a combination of geological factors and agricultural practices. This study highlights the need for further investigation into the potential health implications of consuming these vegetables in the aforementioned region.

Contamination, speciation, and health risk assessment of arsenic in leafy vegetables in Ambagarh Chowki (India).

Green leafy vegetables are essential for a balanced diet, providing vital nutrients for overall well-being. However, concerns arise due to contamination with toxic substances, such as arsenic, posing risks to food safety and human health. This study analyzes inorganic (iAs), monomethyl (MMA), and dimethyl arsenic (DMA) in specific leafy vegetables (Amaranthus tricolor L., Corchorus olitorius L., Cordia myxa L., Hibiscus sabdariffa L., Ipomoea batatas (L.) Lam., Moringa oleifera Lam., and Spinacia oleracea L.) grown in the heavily polluted Ambagarh Chouki region, Chhattisgarh, India. Concentrations of DMA, MMA, and iAs ranged from 0 to 155, 0 to 7, and 131 to 3579 mg·kg-1, respectively. The health quotient (HQ) for iAs ranged between 0.37 and 3.78, with an average value of 2.58 ± 1.08.

Design and Fabrication of Broad-Spectrum Antimicrobial Porous Metallo-Polymeric Microsphere for Water Disinfection.

An expedient and efficient approach is used to synthesize a new class of metallo-polymeric microspheres (MPMs) as antimicrobials to succumb the wide range of bacteria from water. Three types of MPMs, that is, poly[Silver (I)-methacrylate-co-methylmethacrylate] (pAgMA), poly[Copper (II)-methacrylate-co-methyl methacrylate] (pCuMA), and poly[Nickel (II)-methacrylate-co-methylmethacrylate] (pNiMA), are prepared via radical suspension polymerization technique in 3D shape with porous texture. The structural and morphological characterization of the prepared microspheres are examined by analytical techniques. The antimicrobial potentialities of prepared MPMs are investigated at the laboratory scale study, revealing that the MPMs exhibit strong antibacterial activity (≈99.9% killing) against Gram-negative and Gram-positive bacteria [Enterobacter hormaechei (EH), Bacillus megatarium (BM), and Bacillus bataviensis (BB)]. The MacConkey agar medium test reveals that MPMs have substantial biocidal efficacy against broad-spectrum Gram-negative bacteria present in tap water. The MPMs exhibit significant antimicrobial efficacy via contact killing owe to the presence of integrated biocidal metal moiety, which represents that the MPMs are safe for water disinfection.

Construction of arsenic selective chelating resin with iron precursor for removal of low-concentration arsenic: Breakthrough modeling and field deployment.

The presence of exorbitant arsenic contamination in the aquatic environment causes astronomically immense health quandaries affecting millions of people, which may lead to death in the case of prolonged indigestion of arsenic-containing drinking water. Herein, we are reporting porous chelating resin with an iron precursor for the removal of arsenic ions from water. Weak acid cation resin was functionalized under varying experimental conditions to get a suitable resin with high arsenic uptake. The theoretical results revealed that the maximum Langmuir adsorption capacities of 3.27 mg g-1 and 1.13 mg g-1 were achieved for As(V) and As(III), respectively. The kinetics of adsorption followed the pseudo-second-order (PSO) model with a high determination coefficient (R2) of 0.9963 and 0.9895 for As(V) and As(III), respectively. The Adams-Bohart, Thomas, Yoon-Nelson, and Pore diffusion models were used to identify the breakthrough curve in the fixed bed adsorption column. The column performance improved with a larger bed height (55 cm), low concentration of influent (0.25 mg L-1), and low flow rate of influent (80 mL min-1). Under this condition, the breakthrough time and exhaustion time were 314 min and 408 min for As(V) and 124 min and 185 min for As(III), respectively.

Different Efflux Pump Systems in Acinetobacter baumannii and Their Role in Multidrug Resistance.

Several infections, such as pneumonia, urinary tract infections (UTIs), as well as bloodstream, skin, and soft tissue infections, are caused by Acinetobacter baumannii, a nosocomial pathogen and Gram-negative coccobacillus. Due to its resistance to a variety of medications, multidrug therapy, and occasionally pan therapies, this bacterium is a huge public health concern. Drug resistance is a big worry not only in A. baumannii, but it is also a major challenge in many other diseases. Antibiotic resistance, biofilm development, and genetic alterations are all linked to variables like the efflux pump. Efflux pumps are transport proteins involved in the extrusion of hazardous substrates from within cells into the external environment (including nearly all types of therapeutically relevant antibiotics). Both Gram-positive and Gram-negative bacteria, as well as eukaryotic organisms, contain these proteins. Efflux pumps may be specialized for a single substrate or can transport a variety of structurally dissimilar molecules (including antibiotics of many classes); these pumps have been linked to multiple drug resistance (MDR). There are five primary families of efflux transporters in the prokaryotic kingdom: MF (major facilitator), MATE (multidrug and toxic efflux), RND (resistance-nodulation-division), SMR (small multidrug resistance), and ABC (ATP-binding cassette). The efflux pumps and their types as well as the mechanisms of an efflux pump involved in multidrug resistance in bacteria have been discussed here. The main focus is on the variety of efflux pumps commonly found in A. baumannii, along with their mechanism by which they make this bacteria drug resistant. The efflux-pump-inhibitor-based strategies that are significant in targeting efflux pumps in A. baumannii have also been discussed. The connection of biofilm and bacteriophage with the efflux pump can prove as an efficient strategy for targeting efflux-pump-based resistance in A. baumannii.

Nerve fibre morphometry with transmission electron microscopy: Application of the nucleator probe in ImageJ.

Stereology and semiautomated binary image histomorphometry are two common methods used for morphometry of nerve fibres. Nucleator probe can be used for the estimation of morphometric parameters like diameter, perimeter, area and volume of a structure that is approximately either a circle or a sphere. In this study, we estimated these parameters with the help of ImageJ software on calibrated transmission electron micrographs. We procured samples of the cochlear nerve (CN) during winter months, within 6-12 hours of death, to reduce post-mortem autolytic changes. The temporal bones containing the CN were fixed by immersion in chilled paraformaldehyde. After dissecting out from the petrous part of the temporal bone, the CN were osmicated and processed for embedding in resin. From the resin blocks, silver coloured (70 nm) ultrathin sections were cut and picked on 300-mesh copper grids, stained with uranyl acetate and lead citrate and viewed under Tecnai G2-20 transmission electron microscope. The transmission electron micrographs had scale bars embedded into them by the software at the time of imaging, and the morphometric parameters of randomly selected nerve fibres were measured using the ImageJ software. The ImageJ software could become a low-cost and dependable tool for nerve fibre morphometry.•Nucleator probe is used for the estimation of morphometric parameters like diameter, perimeter, area or volume•Morphometric parameters were estimated by the ImageJ software on calibrated transmission electron micrographs•The ImageJ software could become a low-cost and dependable tool for nerve fibre morphometry.

One-pot facile approach to design an efficient macro-porous polymeric matrix to remediate Hg(II)and Pb(II) from aqueous medium and its performance evaluation study by mathematical modelling.

In the present scenario discharge of heavy-metal ions into water bodies is a global threat that is causing serious health hazards even in low concentrations. Thus, in order to remediate the heavy-metal [Hg(II) and Pb(II)] toxicity, an organic-inorganic hybrid functional porous metallo-polymeric network i. e, poly(Zirconyl methacrylate-co-1-vinyl imidazole) (pZrVIm) was fabricated via one-pot facile synthesis approach. The pZrVIm architecture has shown high removal efficiency for Hg(II) and Pb(II) aqueous medium even in extremely low quantities. Advanced instrumental techniques were used to characterize the structural and morphological characteristics of pZrVIm. Different experimental variables i.e., reaction time, pH, initial feed concentration, co-ion effects etc. were explored to examine adsorption behaviour. The maximum adsorption capacities (qmax) of pZrVIm5 were calculated as 168.06 and 162.34 mg g-1 for Hg(II) and Pb(II) respectively by the Langmuir isotherm model. Data from isotherms showed that monolayer adsorption on a homogeneous surface is the rate-limiting stage and followed pseudo-second-order kinetic process. The Artificial Neural Network (ANN) modelling was used to validate kinetics and isotherm data which revealed high accuracy of the model with correlation coefficient values (R = 0.99). Various types of isotherm models such as Langmuir, Freundlich, Dubinin-Radushkevich, Temkin, Redlich-Peterson, Toth and Koble-Corigen have been studied to determine the adsorption phenomena. The pore diffusion model revealed breakthrough time of 91 h and 84 h, Hg(II) and Pb(II) with the feed concentration of 15 mg L-1 respectively. The study revealed that pZrVIm5 has great potential for heavy metal ions remediation for water treatment.

Reverse vaccinology approach to design a vaccine targeting membrane lipoproteins of Salmonella typhi.

Typhoid fever caused by Salmonella is one of the major health issues worldwide, resulting in millions of cases and has very high rates of morbidities. The therapeutic approaches need to be updated for the effective elimination of the bacterial pathogen. The designing of the multiepitope vaccine against Salmonella using comparative proteomics and reverse vaccinology has covered up all the epitopes that induce sufficient immune responses in the host body. Out of the 4293 proteins, 15 outer membrane proteins have been selected based on their antigenicity, low transmembrane helix (<1), and virulence-associated factors. With the help of the reverse vaccinology approach, the epitopes of MHC Class I, Class II, and B-cell with antigenic, low toxicity, and that have the potential to generate immunogenic response have been identified. Based on the comparative analysis of all the epitopes, a multiepitope-based construct has been designed. Based on physicochemical properties and docking scores for HLA and TLR4, the VC5 construct has been selected, and the molecular dynamic simulation studies have confirmed their interaction. The dissociation constant of the VC5 and TLR4 was found to be 3.1 x 10-9. Different immune cell activation has been analyzed, representing the potentiality of the VC5 construct as an effective vaccine target. In silico cloning of VC5 in pET28a has also been performed, which requires experimental validation. Therefore, the present study designs a multi-epitope vaccine VC5 targeted to the membrane lipoproteins of Salmonella typhi.Communicated by Ramaswamy H. Sarma.

Thin film poly(vinyl alcohol)-polysulfone composite membranes based on recycled polysulfone: salt separation performances.

Poly(vinyl alcohol) (PVA)-coated membranes on polysulfone (Ps) (Memb-Pv) were cross-linked using the dibasic maleic acid. The tailoring of membrane properties (viz. hydrophilicity, permeability) develops through the ester linkage due to cross-linking of PVA and maleic acid (MA). Series of Ps asymmetric membranes were prepared using the successive stages of phase inversion of Ps materials. The recyclability approaches of polysulfone (Ps) pertained. The characteristics and transport properties of all the membranes are evaluated. FTIR-ATR, scanning electron (SEM), and atomic force microscopy (AFM) are used for the structural characterization of the membranes. The salient features of Memb-Pv composite membranes support promising results in desalination. The work aimed to highlight the trade-off between the flux and selectivity of composite membranes' salts (bi-/monovalent) through the recycled Ps matrix. The number of recycling stages influences the salt separation performance. The sulfate rejection differs from Memb-IPv (93.26%) to Memb-IVPv (86.70%) for water-I matrix using 2500 mg/L. The defluoridation potential of the membranes is also convincing. The defluoridation performance of Memb-IPv is 87% for 10 mg/L. A significant dimension is added regarding salt separation performance's dependence on the water matrices' nature. The decrease in fluoride separation is ~ 1-3% for the water-2 matrix as the TDS increases.

Kinetics of Severe Acute Respiratory Syndrome Corona Virus-2 Specific Antibody in Corona Virus Diease 2019.

BACKGROUND: Severe Acute Respiratory Syndrome Corona Virus 2019, a novel coronavirus first reported from China in 2019, is the causative agent of pandemic corona virus disease 2019. Antibody response and its dynamics may provide information about natural immunity conferred upon by corona virus disease 2019. METHODS: A health-center-based follow up study of confirmed Severe Acute Respiratory Syndrome Corona Virus-2 infected patients was conducted from December 2020 to June 2021. Patients were followed up to period of 28 weeks during the study. An electrochemiluminescence immunoassay was used to test antibodies elicited by Severe Acute Respiratory Syndrome Corona Virus-2. Socio-demographic and clinical information was collected from each patient. RESULTS: A total of 40 patients (18 males and 22 females) were enrolled in the study, with 90 % seropositivity of SARS-CoV-2 antibody. Antibody level was tracked up to 28 weeks following the infection, and persistence was observed till the end. Antibody level peaked on the 3rd to 4th month, after symptom onset. The male population was found to have higher antibody levels compared to females. Age-wise trend analysis showed lower antibody levels in the younger people (15-30 years) than those older (31-60 years). CONCLUSIONS: We demonstrated that Severe Acute Respiratory Syndrome Corona Virus-2 specific antibodies in corona virus disease 2019 patients persist for at least 28 weeks, peaking at 13 to 20 weeks. Statistically, there was no correlation of antibody levels with the age and sex of individuals. Further study on a larger population is needed for determining long-term immunity.

Synchronous Removal of Arsenic and Fluoride from Aqueous Solution: A Facile Approach to Fabricate Novel Functional Metallopolymer Microspheres.

Concurrence of arsenic (As) and fluoride (F-) ions in groundwater is a serious concern due to their fatal effects. Herein, an attempt was made to fabricate quaternized poly(zirconyl dimethacrylate-co-vinylbenzyl chloride)] (ZrVBZ), a metallopolymeric microsphere in three-dimensional shape with a porous texture. The synthesized ZrVBZ was utilized for the synchronal removal of As and F- from water. Techniques such as Fourier transform infrared spectroscopy, 13C-nuclear magnetic resonance, scanning electron microscopy, and Brunauer-Emmett-Teller surface area were used to characterize the ZrVBZ. The maximum adsorption capacity of ZrVBZ for both fluoride and arsenic (q max F-: 116.5 mg g-1, q max As(V): 7.0 mg g-1, and q max As(III): 6.5 mg g-1) at given experimental conditions (adsorbents' dose: 0.250 g L-1, feed of F-: 50 mg L-1, As(V)/As(III): 2000 µg L-1, and pH: 7.0 ± 0.2) was ascribed to the porous spherical architecture with dual functional sites to facilitate adsorption. The adsorption followed pseudo-second-order kinetics with a correlation coefficient of 0.996, 0.997, and 0.990 for F-, As(V), and As(III), respectively. The isotherm data fitted to the Langmuir isotherm model, and the maximum capacity was 121.5, 7.246, and 6.68 mg g-1 for F-, As(V), and As(III), respectively. The results of this study indicated that ZrVBZ could be used as an effective adsorbent for the simultaneous removal of F-, As(V), and As(III) from an aqueous medium.

The ultrastructural study of human cochlear nerve at different ages.

Ultrastructural and molecular changes in the myelin of the cochlear nerve (CN) have been associated with decreased hearing-acuity with increasing age. But most of these are animal studies or with very few human samples. Hence, we studied the ultrastructure of the human CN at different ages. We obtained samples of CN from persons, who at the time of death belonged to young, middle or old age-groups; defined as ≤ 30, 31 to 50, and ≥ 51 years of age, respectively. These were processed for viewing under a transmission electron microscope (TEM). Morphology and morphometry were assessed after blinding the observer. Measurements of diameter (whole nerve fibre, axon), myelin thickness and calculation of G-ratio were made on calibrated images using ImageJ software. K-Means cluster analysis was performed based on total and inner nerve fibre area. Middle and old age CN showed degenerating axons, splitting of myelin sheath and myelin balloons. Between the middle and old age groups there was significant decrease in axon diameter (p<0.001), inner nerve fibre area (p<0.001), myelin thickness (p<0.001), nerve fibre diameter (p<0.001), and G-ratio (p<0.001). By clustering, we identified three distinct populations of myelinated nerve fibres: large, medium and small. The large fibres (by size), seen in the young, disappeared in the old age-group. We were unable to find any unmyelinated nerve fibres in this study. The morphological deterioration CN fibres may be a visible sign of molecular degeneration and contribute to decreased hearing-acuity.

Antipersister strategies against stress induced bacterial persistence.

The increase in antibiotic non-responsive bacteria is the leading concern in current research oriented to eliminate pathogens. Nowadays, the excess use of antibiotics without specifically understanding the potentiality of killing pathogens and bacterial survival patterns has helped bacteria emerge indefatigably. Bacteria use various mechanisms such as resistance, persistence, and tolerance to ensure survival. Among these, persistence is a mechanism by which bacteria reside in their dormant state, bypassing the effects of treatments, making it crucial for bacterial survival. Persistent bacterial cells arise from the normal bacterial population as a slow-growing subset of bacteria with no metabolic flux. This behavior renders it to survive for a longer duration and at higher concentrations of antibiotics. They are one of the underlying causes of recurrence of bacterial infections. The present article explains the detailed molecular mechanisms and strategies of bacterial persistence, including the toxin-antitoxin modules, DNA damage, the formation of inactive ribosomal complexes, (p)ppGpp network, antibiotic-induced persistence, which are triggered by drug-induced stress. The article also comprehensively covers the epigenetic memory of persistence in bacteria, and anti-persistent therapeutics like antimicrobial molecules, synthetic peptides, acyldepsipeptide antibiotics, and endolysin therapy to reduce persister cell formation and control their frequency. These strategies could be utilized in combating the pathogenic bacteria undergoing persistence.

Implementation of Blended Learning Approach for Improving Anatomy Lectures of Phase I MBBS Students - Learner Satisfaction Survey.

PURPOSE: The application of blended learning approach improves student engagement, active learning and achievement of higher order analytical thinking. Despite that there is still no clear proof that use of computer assisted learning is better than traditional teaching in human anatomy. In this study, a model of collaborative learning strategy is applied, using both online and face-to-face instruction delivery, for the Phase I medical students. The study aimed to innovate a model of blended learning in delivery of gross anatomy lectures and determine the effectiveness of this strategy in comparison to traditional classroom teaching by student's perception analysis. METHODS: Students were provided with PowerPoint lectures, clinical video of patient cases, and lecture summary handouts before the interactive face-to-face lecture session. Learner satisfaction survey was done to validate the student's perception. RESULTS: The students' participation in the survey was 90.4%, with the mean of students' responses on the survey scale was 4.10 ± 0.94 (standard deviation). Highest agreement was 83.1% on the effectiveness of online resources. Students gave minimum agreement of 54.9% about the time allotted for the online materials. Thematic analysis of the perception of student feedback provided with many positive outcomes for this approach. CONCLUSION: We propose that blended learning is a process that helps in enhancing student involvement and participation, motivation for self-directed learning and promotes deeper learning of educational outcomes.

Quality Assessment of an Antimicrobial Resistance Surveillance System in a Province of Nepal.

Antimicrobial resistance (AMR) is a global problem, and Nepal is no exception. Countries are expected to report annually to the World Health Organization on their AMR surveillance progress through a Global Antimicrobial Resistance Surveillance System, in which Nepal enrolled in 2017. We assessed the quality of AMR surveillance data during 2019-2020 at nine surveillance sites in Province 3 of Nepal for completeness, consistency, and timeliness and examined barriers for non-reporting sites. Here, we present the results of this cross-sectional descriptive study of secondary AMR data from five reporting sites and barriers identified through a structured questionnaire completed by representatives at the five reporting and four non-reporting sites. Among the 1584 records from the reporting sites assessed for consistency and completeness, 77-92% were consistent and 88-100% were complete, with inter-site variation. Data from two sites were received by the 15th day of the following month, whereas receipt was delayed by a mean of 175 days at three other sites. All four non-reporting sites lacked dedicated data personnel, and two lacked computers. The AMR surveillance data collection process needs improvement in completeness, consistency, and timeliness. Non-reporting sites need support to meet the specific requirements for data compilation and sharing.

Fetus in Fetu in an Adult Female and Brief Review of Literature.

Fetus in fetu (FIF) is a very rare condition in which malformed fetus is found within the body of a living twin, most commonly within the retroperitoneum. It is a parasitic fetal twin of a diamniotic, monozygotic type. It should be differentiated from teratoma by the presence of organized vertebral column and appropriately arranged other organs or limbs around it. There is no such axial arrangement in teratoma, which has also got definite malignant potential. We report a case of FIF in a 21-year-old lady who presented late with nonspecific abdominal symptoms. Preoperative diagnosis of FIF in this case was made on computed tomography, and the patient underwent exploratory laparotomy with complete excision of mass. The excised mass in a sac was proven to be FIF on the basis of gross and histopathological examination. Surgical excision is the ideal treatment even teratoma being the differential diagnosis.

Scavenging fluoride from the aqueous system with porous organometallic three-dimensional architecture: An emerging adsorbent.

To provide safe water to the suffering community, a porous, and three-dimensional architecture (ZrLMA) is presented for the removal of fluoride from contaminated water. The structural moiety of ZrLMA contains zirconium dimethacrylate (ZrDMA) and lauryl methacrylate (LMA). Various experimental factors, i.e., the effect of adsorbent's dose, feed concentration of fluoride, pH, pHpzc, and interfering ions, are investigated to evaluate its performance. The binding energy between ZrLMA and F- ions is investigated by density functional theory and found to be - 271.3 kJ mol-1, which indicates a high level of interaction between ZrLMA and F- ions at atomic and molecular levels. Freundlich and Langmuir adsorption isotherm models are best fitted with the obtained experimental data with the maximum adsorption capacity for fluoride as 19.8 mg g-1 (pH 7.0 ± 0.2, initial F- concentration: 10 mg l-1 and dose: 0.5 g l-1). The kinetics and thermodynamic parameters (ΔG, ΔH, and ΔS) are also investigated. The uniqueness of the adsorbent is due to its covalently co-ordinated metallic-polymeric moiety, which provides stable architecture during the sorption process irrespective of the nature of the surrounding medium. Due to its absolute structural integrity, the adsorbent does not leach out any trace elements (Zr, F-) in the treated water during the adsorption process while maintaining original characterstics of the water from field water samples as well. An attempt has been made to recover fluoride at the end of the process. Hence, the approach is environment friendly for the remediation and recovery of pollutants due to its excellent reusability with effluent treatment.

Therapeutic strategies against autophagic escape by pathogenic bacteria.

Growing multidrug-resistant (MDR) strains of various infectious bacterial species are hindering research aiming to eliminate such infections. During a bacterial infection, the host response eliminates the pathogen via fusion of the endocytic vesicles with lysosomes, called xenophagy. However, MDR bacteria have evolved strategies to escape xenophagy. In this review, we propose novel therapeutics for overcoming such escape, including chimeric antibiotics, nanoformulations for the induction of autophagy in infected cells, and small interfering (si)RNA-mediated silencing of genes to inhibit the host-pathogen interaction. We also discuss the role of combinations of antibiotics showing synergy, the administrative routes of differentially capped nanoparticles (NPs), and the use of different types of nanoformulations for eliminating pathogenic bacteria from the host.

Establishment of reference range of CD4 T-lymphocyte in healthy Nepalese adults.

OBJECTIVE: CD4 T lymphocytes are the most widely used cellular markers to assess the course of HIV infection, clinical staging and, monitoring the effect of antiretroviral therapy. The regional reference range for Eastern, Central and Western development region of Nepal had already been established whereas the same was still lacking in Mid-western and Far-western development region. The objective of this study was to establish reference range of CD4 T lymphocyte in the remaining two development regions and finally the national reference range using data from previous study. RESULTS: The average values (mean ± SD) of CD4 and CD3 T cell in present study was (819 ± 294) cells/µl and (1546 ± 532) cells/µl, respectively. The absolute CD4 T cell (914 ± 303) and CD3 T cell (1671 ± 560) count in female were significantly higher than those from male, CD4 (757 ± 270) and CD3 (1465 ± 499) (p value-0.000). National reference value of CD4 was determined to be (798 ± 335) cells/µl for healthy Nepalese adults.