Developing Nepal's medicines pricing policy: evidence synthesis and stakeholders' consultation.

Objectives: The objectives of this paper are to (a) explore stakeholders' opinions regarding Nepal's existing medicines pricing practices/situation and (b) build and present a set of medicines pricing policies for Nepal. Methods: A review of the literature and field visits to community retail pharmacies, hospital pharmacies, wholesalers, and distributor outlets in Kathmandu were conducted to assess the medicines pricing situation. Following the literature review, preliminary meetings with stakeholders and field visits were held and a draft interview guide was prepared. Consultative sessions subsequently were undertaken in Kathmandu, Nepal, in January 2023 with representatives from the Department of Drug Administration, Ministry of Health and Population, Association of Pharmaceutical Producers of Nepal, consumer groups, Transparency International, Medicines Importers Association of Nepal/ Pharmaceutical Distributors Association of Nepal, Nepal Chemist and Druggist Association, and Nepal Pharmaceutical Association. Notes were taken during these meetings regarding issues and concerns raised as well as experiences and recommendations for the future, as outlined in the interview guide. Results: The stakeholders in general stated that they do not have any objection to price regulation; however, they believe such regulation should be subject to periodic review. Both the importers and the Ministry of Health and Population have the view that an independent body/authority should be charged with regulating the prices of medicines. A set of policy options to be considered for use in Nepal include cost-plus pricing, external price referencing, internal reference pricing, and mark-up regulations. Conclusion: Key issues related to pricing were identified and suggest that a set of pricing policies and updated regulations need to be considered to establish changes that are transparent, rational, and acceptable to the related stakeholders. Hence, suggestions made in this paper could be useful to inform a rational and fair pricing structure and to improve access to medicines.

Rational Primer and Probe Construction in PCR-Based Assays for the Efficient Diagnosis of Drifting Variants of SARS-CoV-2.

The genome sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been evolving via genomic drifts resulting in "emerging/drifting variants" circulating worldwide. The construction of polymerase chain reaction (PCR) assays for the reliable, efficient, and specific diagnosis of the drifting variants of SARS-CoV-2 is specifically governed by the selection and construction of primers and probes. The efficiency of molecular diagnosis is impacted by the identity/homology of the genome sequence of SARS-CoV-2 with other coronaviruses, drifting variants or variants of concern (VOCs) circulating in communities, inherent capacity of mutation(s) of various target genes of SARS-CoV-2, and concentration of genes of interest in host cells. The precise amplicon selection and construction of primers and probes for PCR-based assays can efficiently discriminate specific SARS-CoV-2 drifting variants. The construction of single nucleotide polymorphism (SNP)-specific primers and probes for PCR assays is pivotal to specifically distinguish SARS-CoV-2 variants present in the communities and contributes to better diagnosis and prevention of the ongoing COVID-19 pandemic. In this study, we have utilized in silico-based bioinformatic tools where the alignment for genes, the positions and types of SNPs/mutations of VOCs, and the relative number of SNPs per nucleotide in different genomic regions were investigated. Optimal and specific genome region (amplicon) selection with comparatively lower mutability in the SARS-CoV-2 genome should be prioritized to design/construct PCR assays for reliable and consistent diagnosis in various regions of the world for a longer duration of time. Further, the rational selection of target genes that is at an optimal detectable concentration in biological samples can bolster PCR assays of high analytical sensitivity. Hence, the construction of primers and probes with the rational selection of targeting specific E gene, genomic regions with highly conserved sequences, multiple target genes with relatively lower mutability and detectable level of concentration, SNP-specific binding regions of spike (S gene) protein, and shorter amplicon size (100-150 bp) are vital for the PCR assays to achieve optimal efficiency in the point-of-care laboratory diagnosis of circulating drifting variants of SARS-CoV-2 with optimal accuracy.

Invasive Aspergillus niger Is the Sole Etiological Agent for CSOM : A Clinical Case from Nepal.

Aspergillus causing chronic suppurative otitis media (CSOM) is rare in immunocompetent people; however, it can occur as a significant opportunistic pathogen in immunocompromised patients. Here, in our study, a 53-year-old diabetic patient having a history of CSOM visited the Department of Otorhinolaryngology-Head and Neck Surgery (ENT-HNS), Tribhuvan University and Teaching Hospital (TUTH), Nepal, in March 2016. Although he was on medication with an antibacterial ear drop from the last 10 days, his right ear was presented with otorrhea, pruritus, otalgia, aural fullness, hearing impairment, and tinnitus from the last 3-4 months. Preliminarily, otoscopy of the right ear revealed the presence of fungal mass. For further diagnosis, ear discharge was aseptically collected and sent to the laboratory to confirm the etiological agents. Findings of laboratory analysis indicated that Gram staining of aural discharge displayed pus cells with fungal spores but did not exhibit bacteria. Furthermore, potassium hydroxide (KOH) mount revealed the presence of fungal spores and septate hyphae with the characteristic of dichotomous branching. Culture in four different bacterial media (chocolate agar, blood agar, MacConkey agar, and Robertson's cooked meat medium) has unveiled no bacterial growth. However, fungal growth was observed in both bacterial and fungal media. Thereafter, the fungal colony was investigated via a lactophenol cotton blue (LPCB) tease mount which displayed the structure of Aspergillus. Aspergillus niger was microbially conformed by specifically characterizing the specific phenotypic biseriate structure of phialides and the black-coloured conidia. For medication, the patient was treated with Candid Ear Drop with clotrimazole (1% w/v) plus lidocaine (2% w/v) for 4 weeks which successfully improved his condition.

An individual randomised efficacy trial of autologous blood products, leukocyte and platelet-rich fibrin (L-PRF), to promote ulcer healing in leprosy in Nepal: the TABLE trial protocol.

BACKGROUND: Leprosy is curable with multidrug therapy and treatment in the early stages can prevent disability. However, local nerve damage can lead to injury and consequently recurring and disfiguring ulcers. The aim of this study is to evaluate the treatment of leprosy ulcers using an autologous blood product; leukocyte and platelet-rich fibrin (L-PRF) to promote healing. METHODS: This is a single-centre study in the Anandaban Hospital, The Leprosy Mission Nepal, Kathmandu, Nepal. Consenting patients (n=130) will be individually randomised in a single-blinded, controlled trial. Participants will be 18 years of age or older, admitted to the hospital with a clean, dry and infection-free chronic foot ulcer between 2 and 20 cm2 in size. If the ulcer is infected, it will be treated before enrolment into the study. The intervention involves the application of leukocyte and platelet-rich fibrin (L-PRF) matrix on the ulcer beds during twice-weekly dressing changes. Controls receive usual care in the form of saline dressings only during their twice-weekly dressing changes. Primary outcomes are the rate of healing assessed using standardised photographs by observers blind to allocated treatment, and time to complete re-epithelialization. Follow-up is at 6 months from randomisation. DISCUSSION: This research will provide valuable information on the clinical and cost-effectiveness of L-PRF in the treatment of leprosy ulcers. An additional benefit is the evaluation of the effects of treatment on quality of life for people living with leprosy ulcers. The results will improve our understanding of the scalability of this treatment across low-income countries for ulcer healing in leprosy and potentially other conditions such as diabetic ulcers. TRIAL REGISTRATION: ClinicalTrials.gov ISRCTN14933421 . Registered on 16 June 2020.

Apt (Adenine Phosphoribosyltransferase) Mutation in Laboratory-Selected Vancomycin-Intermediate Staphylococcus aureus.

Comparative genomic sequencing of laboratory-derived vancomycin-intermediate Staphylococcusaureus (VISA) (MM66-3 and MM66-4) revealed unique mutations in both MM66-3 (in apt and ssaA6), and MM66-4 (in apt and walK), compared to hetero-VISA parent strain MM66. Transcriptional profiling revealed that both MM66 VISA shared 79 upregulated genes and eight downregulated genes. Of these, 30.4% of the upregulated genes were associated with the cell envelope, whereas 75% of the downregulated genes were associated with virulence. In concordance with mutations and transcriptome alterations, both VISA strains demonstrated reduced autolysis, reduced growth in the presence of salt and reduced virulence factor activity. In addition to mutations in genes linked to cell wall metabolism (ssaA6 and walK), the same mutation in apt which encodes adenine phosphoribosyltransferase, was confirmed in both MM66 VISA. Apt plays a role in both adenine metabolism and accumulation and both MM66 VISA grew better than MM66 in the presence of adenine or 2-fluoroadenine indicating a reduction in the accumulation of these growth inhibiting compounds in the VISA strains. MM66 apt mutants isolated via 2-fluoroadenine selection also demonstrated reduced susceptibility to the cell wall lytic dye Congo red and vancomycin. Finding that apt mutations contribute to reduced vancomycin susceptibility once again suggests a role for altered purine metabolism in a VISA mechanism.

Vulnerabilities for Exposure to Emerging Infectious Disease at Urban Settlements in Nepal.

In Nepal, rapid urbanization and rural-to-urban migration especially due to internal civil conflict have catalyzed the development of temporary settlements, often along rivers on undeveloped land. This study conducted surveillance for viruses in small mammals and assessed potential risks for virus transmission to people in urban settlements along rivers in Kathmandu, Nepal. We collected samples from 411 small mammals (100 rodents and 311 shrews) at four riverside settlement sites and detected six viruses from four virus families including Thottapalayam virus; a strain of murine coronavirus; two new paramyxoviruses; and two new rhabdoviruses. Additionally, we conducted surveys of 264 residents to characterize animal-human contact. Forty-eight percent of individuals reported contact with wildlife, primarily with rodents and shrews (91%). Our findings confirm that rodents and shrews should be considered a health threat for residents of temporary settlements, and that assessment of disease transmission risk coupled with targeted surveillance for emerging pathogens could lead to improved disease control and health security for urban populations. Additionally, interventions focused on disease prevention should consider the unique urban ecology and social dynamics in temporary settlements, along with the importance of community engagement for identifying solutions that address specific multi-dimensional challenges that life on the urban river margins presents.

Antimicrobial drug resistant non-typhoidal Salmonella enterica in commercial poultry value chain in Chitwan, Nepal.

BACKGROUND: Antimicrobial resistance (AMR) among bacterial pathogens is a fast-growing public health concern. AMR in non-typhoidal Salmonella serovars (NTS) among food animals is of special concern as this may transmit resistant pathogens to humans during handling or consumption of animal products. In Nepal, the possibility of AMR Salmonella serovars among food animals is an important area of research, particularly in light of the rapidly growing poultry industry, lack of surveillance and proper biosecurity measures; and paucity of relevant data. This study was conducted with the aim to estimate the burden of NTS and associated antimicrobial resistance in the environments of commercial poultry farms and the poultry carcasses in slaughter house. This study also intends to find some basic knowledge of the poultry farmers and their practice relating to the use of antimicrobials, vaccination and biosecurity measures. METHODS: Taking one health approach, a cross-sectional study was carried out in Chitwan district of Nepal between May and October 2017. Various environmental samples viz. farm litter, feed, water, poultry faeces, vehicle swabs, farm swabs from 12 broiler poultry farms and various sections of poultry carcasses from 21 slaughter houses were aseptically collected. These were microbiologically assessed for the presence of NTS serovars and their phenotypic and genotypic indicators of antimicrobial resistance. The poultry farmers were also briefly interviewed regarding their basic biosecurity related knowledge and practices before collecting the environmental samples. RESULTS: Overall, of total environmental samples collected, 50% (31/62) tested positive for NTS serovars with environmental swabs (70%, 8/12) being the most culture positive sample types. Similarly, of 159 tissue samples collected from 24 carcasses, 79% (126/159) were culture positive for NTS serovars. Nearly 97% (153/157) of isolates showed antimicrobial resistance to tetracycline, while 11% (17/157) to ciprofloxacin and 5% (8/157) of isolates were resistant against azithromycin. All 157 isolates were sensitive to meropenem. In terms of AMR genes, tetA (83%, 131/157), QrnS (40%,64/157), mefA (8%, 13/157) and VIM-1 (0.6%, 1/157) were detected in the isolates that corresponded to the AMR to tetracycline, ciprofloxacin, azithromycin and meropenem respectively. In farmers interview, only 42% (5/12) of farmers mentioned of using basic biosecurity measures such as applying lime powder around the farm; 84% (10/12) of farmers reported vaccinating their birds with some vaccine and 75% (9/12) of farmers used various antimicrobials prophylactically such as neomycin (33%, 4/12), colistin (33%, 4/12), furaltadone (33%, 4/12), doxycycline (25%, 3/12), sulfatrimethoprim (25%, 3/12) and tylosin (16%, 2/12). CONCLUSIONS: This study revealed gross contamination of farm environment and subsequent poultry meat samples with NTS serovars that were resistant to several clinically important antimicrobials. Further, inadequacy of even basic biosecurity measures and frequent prophylactic use of antimicrobials in the commercial poultry farms was observed. This reinforces an urgent need to raise awareness and implement proper biosecurity approaches from farms to slaughter houses in order to reduce the burden of NTS contamination of surrounding environment and poultry products. Further, high prevalence AMR among NTS isolates also underscores the need to strengthen the policies to prevent the rampant use of clinically used human antimicrobials in poultry sector.

Species, sex and geo-location identification of seized tiger (Panthera tigris tigris) parts in Nepal-A molecular forensic approach.

Tiger (Panthera tigris) populations are in danger across their entire range due to habitat loss, poaching and the demand for tiger parts. The Bengal tiger (Panthera tigris tigris) is an endangered apex predator with a population size estimated to be less than 200 in Nepal. In spite of strict wildlife protection laws, illegal trade of tiger parts is increasing; and Nepal has become one of the major sources and transit routes for poached wildlife parts. Identification of wildlife parts is often challenging for law enforcement officials due to inadequate training and lack of available tools. Here, we describe a molecular forensic approach to gain insight into illegally trafficked tiger parts seized across Nepal. We created Nepal's first comprehensive reference genetic database of wild tigers through the Nepal Tiger Genome Project (2011-2013). This database has nuclear DNA microsatellite genotype and sex profiles, including geo-spatial information, of over 60% (n = 120) of the wild tigers of Nepal. We analyzed 15 putative cases of confiscated poached tiger parts and all were confirmed to be of tiger. Ten samples were identified as male and five were female. We determined probable geo-source location for 9 of the 14 samples with 6-8 nuclear DNA microsatellite loci using inferences from four different statistical assignment methods. Six samples were assigned to Bardia National Park and one of these was an exact match to a female tiger previously profiled in our fecal DNA reference database. Two tiger samples were assigned to Shuklaphanta Wildlife Reserve and one to Chitwan National Park. We are unable to definitively assign five tiger samples which could be offspring dispersers or might have come from tiger population outside of Nepal. Our study revealed that the western region, particularly Bardia National Park, is a poaching hotspot for illegal tiger trade in Nepal. We present feasibility of using molecular forensic based evidence to incriminate criminals in a court of law in the fight against wildlife crime.

Stress induction in the bacteria Shewanella oneidensis and Deinococcus radiodurans in response to below-background ionizing radiation.

PURPOSE: The 'Linear no-threshold' (LNT) model predicts that any amount of radiation increases the risk of organisms to accumulate negative effects. Several studies at below background radiation levels (4.5-11.4 nGy h(-1)) show decreased growth rates and an increased susceptibility to oxidative stress. The purpose of our study is to obtain molecular evidence of a stress response in Shewanella oneidensis and Deinococcus radiodurans grown at a gamma dose rate of 0.16 nGy h(-1), about 400 times less than normal background radiation. MATERIALS AND METHODS: Bacteria cultures were grown at a dose rate of 0.16 or 71.3 nGy h(-1) gamma irradiation. Total RNA was extracted from samples at early-exponential and stationary phases for the rt-PCR relative quantification (radiation-deprived treatment/background radiation control) of the stress-related genes katB (catalase), recA (recombinase), oxyR (oxidative stress transcriptional regulator), lexA (SOS regulon transcriptional repressor), dnaK (heat shock protein 70) and SOA0154 (putative heavy metal efflux pump). RESULTS: Deprivation of normal levels of radiation caused a reduction in growth of both bacterial species, accompanied by the upregulation of katB, recA, SOA0154 genes in S. oneidensis and the upregulation of dnaK in D. radiodurans. When cells were returned to background radiation levels, growth rates recovered and the stress response dissipated. CONCLUSIONS: Our results indicate that below-background levels of radiation inhibited growth and elicited a stress response in two species of bacteria, contrary to the LNT model prediction.

The gastrointestinal microbiota and colorectal cancer.

The human gut is home to a complex and diverse microbiota that contributes to the overall homeostasis of the host. Increasingly, the intestinal microbiota is recognized as an important player in human illness such as colorectal cancer (CRC), inflammatory bowel diseases, and obesity. CRC in itself is one of the major causes of cancer mortality in the Western world. The mechanisms by which bacteria contribute to CRC are complex and not fully understood, but increasing evidence suggests a link between the intestinal microbiota and CRC as well as diet and inflammation, which are believed to play a role in carcinogenesis. It is thought that the gut microbiota interact with dietary factors to promote chronic inflammation and CRC through direct influence on host cell physiology, cellular homeostasis, energy regulation, and/or metabolism of xenobiotics. This review provides an overview on the role of commensal gut microbiota in the development of human CRC and explores its association with diet and inflammation.

Gut microbiome and colorectal adenomas.

The trillions of bacteria that naturally reside in the human gut collectively constitute the complex system known the gut microbiome, a vital player for the host's homeostasis and health. However, there is mounting evidence that dysbiosis, a state of pathological imbalance in the gut microbiome is present in many disease states. In this review, we present recent insights concerning the gut microbiome's contribution to the development of colorectal adenomas and the subsequent progression to colorectal cancer (CRC). In the United States alone, CRC is the second leading cause of cancer deaths. As a result, there is a high interest in identifying risk factors for adenomas, which are intermediate precursors to CRC. Recent research on CRC and the microbiome suggest that modulation of the gut bacterial composition and structure may be useful in preventing adenomas and CRC. We highlight the known risk factors for colorectal adenomas and the potential mechanisms by which microbial dysbiosis may contribute to the etiology of CRC. We also underscore novel findings from recent studies on the gut microbiota and colorectal adenomas along with current knowledge gaps. Understanding the microbiome may provide promising new directions towards novel diagnostic tools, biomarkers, and therapeutic interventions for CRC.

The isolation of Staphylococcus aureus tea tree oil-reduced susceptibility mutants.

Tea tree oil (TTO)-reduced susceptibility (TTORS) mutants of two Staphylococcus aureus laboratory strains were isolated utilizing TTO gradient plates. Attempts to isolate TTORS mutants employing agar plates containing single TTO concentrations failed. All TTORS mutants demonstrated a small colony variant (SCV) phenotype and produced cells with a smaller diameter, as determined by scanning electron microscopy. The addition of SCV auxotrophic supplements to media did not lead to an increase in TTORS mutant colony size. Revertants were also isolated from the TTORS mutants following growth in drug-free media, and all revertant strains demonstrated phenotypes similar to their respective parent strains. Transmission electron microscopy revealed that an SH1000 TTORS mutant demonstrated a thinner cell wall and novel septal invaginations compared with parent strain SH1000. In addition, comparative genomic sequencing did not reveal any mutations in an SH1000 TTORS mutant previously linked to well-characterized SCV genotypes. This study demonstrates that TTO can select for a unique SCV phenotype.

Tea tree oil-induced transcriptional alterations in Staphylococcus aureus.

Tea tree oil (TTO) is a steam distillate of Melaleuca alternifolia that demonstrates broad-spectrum antibacterial activity. This study was designed to document how TTO challenge influences the Staphylococcus aureus transcriptome. Overall, bioinformatic analyses (S. aureus microarray meta-database) revealed that both ethanol and TTO induce related transcriptional alterations. TTO challenge led to the down-regulation of genes involved with energy-intensive transcription and translation, and altered the regulation of genes involved with heat shock (e.g. clpC, clpL, ctsR, dnaK, groES, groEL, grpE and hrcA) and cell wall metabolism (e.g. cwrA, isaA, sle1, vraSR and vraX). Inactivation of the heat shock gene dnaK or vraSR which encodes a two-component regulatory system that responds to peptidoglycan biosynthesis inhibition led to an increase in TTO susceptibility which demonstrates a protective role for these genes in the S. aureus TTO response. A gene (mmpL) encoding a putative resistance, nodulation and cell division efflux pump was also highly induced by TTO. The principal antimicrobial TTO terpene, terpinen-4-ol, altered ten genes in a transcriptional direction analogous to TTO. Collectively, this study provides additional insight into the response of a bacterial pathogen to the antimicrobial terpene mixture TTO.