Infectious sources of Histoplasmosis and molecular techniques for its identification

Histoplasmosis, a fungal infection caused by Histoplasma capsulatum (H. capsulatum), acquired from contaminated soil with droppings of chicken or birds and found to be distributed in many parts of the world. The prevalence of histoplasmosis has not well studied in Nepal. The common symptoms of acute and epidemic histoplasmosis include high fever, cough, and asthenia and weight loss. Most of the infections associated with histoplasmosis are asymptomatic. People with compromised immune systems such as HIV/AIDS (PLWHA), cancer, and organ transplant recipients are at risk of developing this disease. In this review, we have summarised the current status of histoplasmosis in Nepal and molecular techniques available for its identification. To date, the significant outbreak is not reported in Nepal, but the risk of infection for the vulnerable population cannot be undermined. Appropriate preventive measures and treatment on time can reduce the burden of this fungal disease. Further, this review is also focused on molecular identification of H. capsulatum. Hence, careful considerations by concerned stakeholders for national surveillance programs and the treatment of patients on time after proper diagnosis is highly recommended

Zooming in on the intracellular microbiome composition of bacterivorous <i>Acanthamoeba</i> isolates

Acanthamoeba, a free-living amoeba in water and soil, is an emerging pathogen causing severe eye infection known as Acanthamoeba keratitis. In its natural environment, Acanthamoeba performs a dual function as an environmental heterotrophic predator and host for a range of microorganisms that resist digestion. Our objective was to characterize the intracellular microorganisms of phylogenetically distinct Acanthamoeba spp. isolated in Australia and India through directly sequencing 16S rRNA amplicons from the amoebae. The presence of intracellular bacteria was further confirmed by in situ hybridization and electron microscopy. Among the 51 isolates assessed, 41% harboured intracellular bacteria which were clustered into four major phyla: Pseudomonadota (previously known as Proteobacteria), Bacteroidota (previously known as Bacteroidetes), Actinomycetota (previously known as Actinobacteria), and Bacillota (previously known as Firmicutes). The linear discriminate analysis effect size analysis identified distinct microbial abundance patterns among the sample types; Pseudomonas species was abundant in Australian corneal isolates (P &lt; 0.007), Enterobacteriales showed higher abundance in Indian corneal isolates (P &lt; 0.017), and Bacteroidota was abundant in Australian water isolates (P &lt; 0.019). The bacterial beta diversity of Acanthamoeba isolates from keratitis patients in India and Australia significantly differed (P &lt; 0.05), while alpha diversity did not vary based on the country of origin or source of isolation (P &gt; 0.05). More diverse intracellular bacteria were identified in water isolates as compared with clinical isolates. Confocal and electron microscopy confirmed the bacterial cells undergoing binary fission within the amoebal host, indicating the presence of viable bacteria. This study sheds light on the possibility of a sympatric lifestyle within Acanthamoeba, thereby emphasizing its crucial role as a bunker and carrier of potential human pathogens

Assessment of genotypes, endosymbionts and clinical characteristics of <i>Acanthamoeba</i> recovered from ocular infection

INTRODUCTION: Acanthamoeba is an emerging pathogen, infamous for its resilience against antiprotozoal compounds, disinfectants and harsh environments. It is known to cause keratitis, a sight-threatening, painful and difficult to treat corneal infection which is often reported among contact lens wearers and patients with ocular trauma. Acanthamoeba comprises over 24 species and currently 23 genotypes (T1-T23) have been identified. AIMS: This retrospective study was designed to examine the Acanthamoeba species and genotypes recovered from patients with Acanthamoeba keratitis (AK), determine the presence of endosymbionts in ocular isolates of Acanthamoeba and review the clinical presentations. METHODOLOGY: Thirteen culture-confirmed AK patients treated in a tertiary eye care facility in Hyderabad, India from February to October 2020 were included in this study. The clinical manifestations, medications and visual outcomes of all patients were obtained from medical records. The Acanthamoeba isolates were identified by sequencing the ribosomal nuclear subunit (rns) gene. Acanthamoeba isolates were assessed for the presence of bacterial or fungal endosymbionts using molecular assays, PCR and fluorescence in situ hybridization (FISH). RESULTS: The mean age of the patients was 33 years (SD ± 17.4; 95% CI 22.5 to 43.5 years). Six (46.2%) cases had AK associated risk factors; four patients had ocular trauma and two were contact lens wearers. A. culbertsoni (6/13, 46.2%) was the most common species, followed by A. polyphaga and A. triangularis. Most of the isolates (12/13) belonged to genotype T4 and one was a T12; three sub-clusters T4A, T4B, and T4F were identified within the T4 genotype. There was no significant association between Acanthamoeba types and clinical outcomes. Eight (61.5%) isolates harboured intracellular bacteria and one contained Malassezia restricta. The presence of intracellular microbes was associated with a higher proportion of stromal infiltrates (88.9%, 8/9), epithelial defect (55.6%, 5/9) and hypopyon (55.6%, 5/9) compared to 50% (2/4), 25% (1/4) and 25% (1/4) AK cases without intracellular microbes, respectively. CONCLUSIONS: Genotype T4 was the predominant isolate in southern India. This is the second report of T12 genotype identified from AK patient in India, which is rarely reported worldwide. The majority of the Acanthamoeba clinical isolates in this study harboured intracellular microbes, which may impact clinical characteristics of AK. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12879-022-07741-4

Data_Sheet_1_Participatory Approach to Develop Evidence-Based Clinical Ethics Guidelines for the Care of COVID-19 Patients: A Mixed Method Study From Nepal

Questionnaire and feedback form produced for the study, "Participatory Approach to Develop Evidence-Based Clinical Ethics Guidelines for the Care of COVID-19 Patients: A Mixed Method Study From Nepal"

A qualitative study to understand drivers of psychoactive substance use among Nepalese youth

Background Psychoactive substance use among youth is an emerging public health issue in Nepal. This exploratory study aimed to better understand the drivers of psychoactive substance use among Nepalese youth in Rupandehi district of Nepal. Materials and methods This study used a qualitative approach for data collection. Both in-depth interviews (IDI, seven participants) and focus group discussions (FGD, 13 participants) were conducted among study participants who self-reported as psychoactive substance users or had history of psychoactive substance use. Participants for IDI were aged between 11 and 24 years and between 18 and 35 years old for FGDs. Semi-structured interview guides were prepared separately for IDIs and FDGs. Interviews were conducted in Nepali language and were audio recorded, which were there transcribed and translated into English for coding and analyses. In addition, interviews notes were taken by two research assistants. An inductive thematic analysis was used to analyze the data. Results This study identified a range of drivers of psychoactive substances use among Nepalese youths. Themes included (i) socio-cultural factors, (ii) individual factors, (iii) academic environment, (iv) physical environment and the (v) influence of media. The socio-cultural factors were categorized into sub-themes of family relationships, ethnic identity and psychoactive substance use and lack of social acceptance. Individual factors included peer pressure, stress relief and coping with financial challenges. Accessibility and availability of psychoactive substances in the surrounding environment and lack of monitoring and reinforcement of rules/ law and regulations were other drivers to psychoactive substance use among this Nepalese youth cohort. Conclusion Our study identified several important drivers of psychoactive substance use among youth in the Rupandehi district of Nepal. Future works are anticipated to further explore youth initiation and use of psychoactive substances and support the design of interventions that address these risk factors to reduce and prevent subsequent harms

Evaluation of Phytochemical, Antioxidant and Antibacterial Activities of Selected Medicinal Plants

Medicinal plants are important reservoirs of bioactive compounds that need to be explored systematically. Because of their chemical diversity, natural products provide limitless possibilities for new drug discovery. This study aimed to investigate the biochemical properties of crude extracts from fifteen Nepalese medicinal plants. The total phenolic contents (TPC), total flavonoid contents (TFC), and antioxidant activity were evaluated through a colorimetric approach while the antibacterial activities were studied through the measurement of the zone of inhibition (ZoI) by agar well diffusion method along with minimum inhibitory concentrations (MIC) by broth dilution method. The methanolic extracts of Acacia catechu and Eupoterium adenophorum showed the highest TPC (55.21 ± 11.09 mg GAE/gm) and TFC (10.23 ± 1.07 mg QE/gm) among the studied plant extracts. Acacia catechu showed effective antioxidant properties with an IC50 value of 1.3 μg/mL, followed by extracts of Myrica esculenta, Syzygium cumini, and Mangifera indica. Morus australis exhibited antibacterial activity against Klebsiella pneumoniae (ZoI: 25mm, MIC: 0.012 mg/mL), Staphylococcus aureus ATCC 25923 (ZoI: 22 mm, MIC: 0.012 mg/mL), Pseudomonas aeruginosa (ZoI; 20 mm, MIC: 0.05 mg/mL), and methicillin-resistant Staphylococcus aureus (MRSA) (ZoI: 19 mm, MIC: 0.19 mg/mL). Morus australis extract showed a broad-spectrum antibacterial activity, followed by Eclipta prostrata, and Hypericum cordifolium. Future study is recommended to explore secondary metabolites of those medicinal plants to uncover further clinical efficacy

Participatory Approach to Develop Evidence-Based Clinical Ethics Guidelines for the Care of COVID-19 Patients: A Mixed Method Study From Nepal.

During health emergencies such as the COVID-19 pandemic, healthcare workers face numerous ethical challenges while catering to the needs of patients in healthcare settings. Although the data recapitulating high-income countries ethics frameworks are available, the challenges faced by clinicians in resource-limited settings of low- and middle-income countries are not discussed widely due to a lack of baseline data or evidence. The Nepali healthcare system, which is chronically understaffed and underequipped, was severely affected by the COVID-19 pandemic in its capacity to manage health services and resources for needy patients, leading to ethical dilemmas and challenges during clinical practice. This study aimed to develop a standard guideline that would address syndemic ethical dilemmas during clinical care of COVID-19 patients who are unable to afford standard-of-care. A mixed method study was conducted between February and June of 2021 in 12 government designated COVID-19 treatment hospitals in central Nepal. The draft guideline was discussed among the key stakeholders in the pandemic response in Nepal. The major ethical dilemmas confronted by the study participants (50 healthcare professionals providing patient care at COVID-19 treatment hospitals) could be grouped into five major pillars of ethical clinical practice: rational allocation of medical resources, updated treatment protocols that guide clinical decisions, standard-of-care regardless of patient's economic status, effective communication among stakeholders for prompt patient care, and external factors such as political and bureaucratic interference affecting ethical practice. This living clinical ethics guideline, which has been developed based on the local evidence and case stories of frontline responders, is expected to inform the policymakers as well as the decision-makers positioned at the concerned government units. These ethics guidelines could be endorsed with revisions by the concerned regulatory authorities for the use during consequent waves of COVID-19 and other epidemics that may occur in the future. Other countries affected by the pandemic could conduct similar studies to explore ethical practices in the local clinical and public health context

Identification and quantification of <i>Acanthamoeba</i> spp. within seawater at four coastal lagoons on the east coast of Australia

Acanthamoeba is an opportunistic free-living heterotrophic protist that is the most predominant amoeba in diverse ecological habitats. Acanthamoeba causes amoebic keratitis (AK), a painful and potentially blinding corneal infection. Major risk factors for AK have been linked to non-optimal contact lens hygiene practices and Acanthamoeba contamination of domestic and recreational water. This study investigated the incidence and seasonal variation of Acanthamoeba spp. within coastal lagoons located on the eastern coast of Australia and then examined the association between Acanthamoeba and water abiotic factors and bacterial species within the water.Water samples were collected from four intermittently closed and open lagoons (ICOLLs) (Wamberal, Terrigal, Avoca and Cockrone) every month between August 2019 to July 2020 except March and April. qPCR was used to target the Acanthamoeba 18S rRNA gene, validated by Sanger sequencing. Water abiotic factors were measured in situ using a multiprobe metre and 16S rRNA sequencing (V3-V4) was performed to characterise bacterial community composition. Network analysis was used to gauge putative associations between Acanthamoeba incidence and bacterial amplicon sequence variants (ASVs).Among 206 water samples analysed, 79 (38.3%) were Acanthamoeba positive and Acanthamoeba level was significantly higher in summer compared with winter, spring, or autumn (p = 0.008). More than 50% (23/45) water samples of Terrigal were positive for Acanthamoeba which is a highly urbanised area with extensive recreational activities while about 32% (16/49) samples were positive from Cockrone that is the least impacted lagoon by urban development. All sequenced strains belonged to the pathogenic genotype T4 clade except two which were of genotype clades T2 and T5. Water turbidity, temperature, intl1 gene concentration, and dissolved O2 were significantly associated with Acanthamoeba incidence (p &lt; 0.05). The ASVs level of cyanobacteria, Pseudomonas spp., Candidatus spp., and marine bacteria of the Actinobacteria phylum and Acanthamoeba 18S rRNA genes were positively correlated (Pearson's r ≥ 0.14). The presence of Acanthamoeba spp. in all lagoons, except Wamberal, was associated with significant differences in the composition of bacterial communities (beta diversity).The results of this study suggest that coastal lagoons, particularly those in urbanised regions with extensive water recreational activities, may pose an elevated risk to human health due to the relatively high incidence of pathogenic Acanthamoeba in the summer. These findings underscore the importance of educating the public about the rare yet devastating impact of AK on vision and quality of life, highlighting the need for collaborative efforts between public health officials and educators to promote awareness and preventive measures, especially focusing lagoons residents and travellers

Perceived fear of COVID-19 and its associated factors among Nepalese older adults in eastern Nepal : a cross-sectional study

Coronavirus disease 2019 (COVID-19) has affected all age groups worldwide, but older adults have been affected greatly with an increased risk of severe illness and mortality. Nepal is struggling with the COVID-19 pandemic. The normal life of older adults, one of the vulnerable populations to COVID-19 infection, has been primarily impacted. The current evidence shows that the COVID-19 virus strains are deadly, and non-compliance to standard protocols can have serious consequences, increasing fear among older adults. This study assessed the perceived fear of COVID-19 and associated factors among older adults in eastern Nepal. Methods A cross-sectional study was conducted between July and September 2020 among 847 older adults (?60 years) residing in three districts of eastern Nepal. Perceived fear of COVID-19 was measured using the seven-item Fear of COVID-19 Scale (FCV-19S). Multivariate logistic regression identified the factors associated with COVID-19 fear. Results The mean score of the FCV-19S was 18.1 (SD = 5.2), and a sizeable proportion of older adults, ranging between 12%-34%, agreed with the seven items of the fear scale. Increasing age, Dalit ethnicity, remoteness to the health facility, and being concerned or overwhelmed with the COVID-19 were associated with greater fear of COVID-19. In contrast, preexisting health conditions were inversely associated with fear. Conclusion Greater fear of the COVID-19 among the older adults in eastern Nepal suggests that during unprecedented times such as the current pandemic, the psychological needs of older adults should be prioritized. Establishing and integrating community-level mental health support as a part of the COVID-19 preparedness and response plan might help to combat COVID-19 fear among them