Patterns and determinants of the global herbivorous mycobiome.

Despite their role in host nutrition, the anaerobic gut fungal (AGF) component of the herbivorous gut microbiome remains poorly characterized. Here, to examine global patterns and determinants of AGF diversity, we generate and analyze an amplicon dataset from 661 fecal samples from 34 mammalian species, 9 families, and 6 continents. We identify 56 novel genera, greatly expanding AGF diversity beyond current estimates (31 genera and candidate genera). Community structure analysis indicates that host phylogenetic affiliation, not domestication status and biogeography, shapes the community rather than. Fungal-host associations are stronger and more specific in hindgut fermenters than in foregut fermenters. Transcriptomics-enabled phylogenomic and molecular clock analyses of 52 strains from 14 genera indicate that most genera with preferences for hindgut hosts evolved earlier (44-58 Mya) than those with preferences for foregut hosts (22-32 Mya). Our results greatly expand the documented scope of AGF diversity and provide an ecologically and evolutionary-grounded model to explain the observed patterns of AGF diversity in extant animal hosts.

Effect of altitude and solvent on Psidium guajava Linn. leaves extracts: phytochemical analysis, antioxidant, cytotoxicity and antimicrobial activity against food spoilage microbes.

BACKGROUND: Guava (Psidium guajava Linn.) has been traditionally used in the treatment of a wide range of diseases due to its rich content of secondary metabolites. AIM: This study was aimed to evaluate the effect of altitude and solvent systems on guava leaves crude extract's phenolics and flavonoid content, antioxidant, antimicrobial, and toxicity nature. METHODS: Guava leaves were collected from three different geographical locations in Nepal while solvents with an increasing polarity index were used for extraction. The yield percentage of extracts was calculated. Total Phenolic Content, Total Flavonoid Content, and antioxidant activity were determined by the Folin-Ciocalteu method, Aluminium chloride colorimetric method, and DPPH (2,2'-Diphenyl-1-picrylhydrazyl) assay respectively. The quantification of fisetin and quercetin was performed using the HPLC with method validation. The antimicrobial activity of the extracts was tested against bacteria and fungus isolated from spoiled fruits and vegetables and identified through 16s and 18s rRNA sequencing. Finally, Brine Shrimp Lethality Assay (BSLA) was used for testing the toxicity of the extracts. RESULTS: The phenolic and total flavonoid content was found to be higher in ethanol extract (331.84 mg GAE/g dry extract) and methanol extract (95.53 mg QE/g dry extract) from Kuleshwor respectively. Water extract of guava leaves from Kuleshwor (WGK) did not show significantly different antioxidant activity when compared to methanol and ethanol extracts. Fisetin and quercetin were higher in WGK (1.176 mg/100 g) and (10.967 mg/100 g) dry extract weight respectively. Antibacterial activity against food spoilage bacteria was dose-dependent and found to be highest for all the extracts from different solvents and altitudes at higher concentrations (80 mg/ml). Similarly, methanol and ethanol guava extracts from all locations showed antifungal activity against Geotrichum candidum RIBB-SCM43 and Geotrichum candidum RIBB-SCM44. WGK was found to be non-toxic. CONCLUSION: Our study concludes that the antioxidant and antimicrobial activity of WGK was found to be similar statistically to that of methanol and ethanol extracts of Bishnupur Katti and Mahajidiya. These results suggest the possibility of using water as a sustainable solvent to extract natural antioxidant and antimicrobial compounds which can further be used as natural preservatives to extend the shelf life of fruits and vegetables.

Isolation and Characterization of Lytic Bacteriophage Against Multi-drug Resistant Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen frequently causing healthcare-associated infections. The apocalyptic rise of antimicrobial resistance has rekindled interest in age-old phage therapy that uses phages (viruses that infect bacteria) to kill the targeted pathogenic bacteria. Because of its specificity, phages are often considered as potential personalized therapeutic candidate for treating bacterial infections. METHODS: In this study, we isolated and purified lytic phages against multi-drug resistant P. aeruginosa using soft agar overlay technique. Phage characteristics like thermal and pH stability, latent period and burst size were determined using one-step growth assay while multiple host range spectrum was determined by spot assay. The phages were further characterized using protein profiling. RESULTS: Three Pseudomonas phages (øCDBT-PA31, øCDBT-PA56 and øCDBT-PA58) were isolated from the holy rivers of Kathmandu valley. Among 3 phages, øCDBT-PA31 demonstrated multiple host range and could lyse multi-drug resistant strain of P. aeruginosa. Further, øCDBT-PA31 showed latent period of 30 minutes with corresponding burst sizes of 423-525 PFU/cell. Interestingly, øCDBT-PA31 also tolerated a wide range of adverse conditions, such as high temperature (50°C) and pH 3-11. Further, protein profiling revealed that øCDBT-PA31 has 4 and øCDBT-PA11 had 3 distinct bands in the gradient gel ranging from approximately 3.5-29 kilodaltons (kDa) suggesting them to be morphologically distinct from each other. CONCLUSIONS: As multi-drug resistant bacteria are emerging as a global problem, lytic phages can be an alternative treatment strategy when all available antibiotics fail.

Serological markers and molecular analysis of hepatitis B infection in a tertiary care hospital at Kathmandu, Nepal.

AIM: To analyze the serology and molecular markers of the hepatitis B-infected patients from the tertiary care hospital at Kathmandu in Nepal. METHODS: A total of 399 blood samples of patients from Sukraraj Tropical and Infectious Disease Hospital, Teku, Kathmandu, were collected. Samples were tested for HBsAg, HBeAg, and IgM anti-HBc using ELISA method. The samples were further categorized as acute and chronic. The genotyping was performed by real-time polymerase chain reaction (real-time PCR) and further validated by sequencing. RESULTS: Out of 399 samples that were collected, 271 and 128 samples were acute and chronic cases respectively. Fifty-six samples were genotyped by qPCR, out of which 40 samples belonged to genotype D, 4 to C/D recombinant, 5 to genotype C, 3 to genotype B, and 4 were genotype A respectively. From these, 15 samples were used for sequencing of P (polymerase) gene and S (surface) genes. Thus, obtained sequences were used to construct neighbor-joining tree using Tamura-Nei model evolution and further validated by Bayesian analysis. A total of four sub-genotypes namely A1, C1, D1, and D5 were detected. CONCLUSION: Hepatitis B virus infection is a global health problem affecting about 257 million people worldwide. In Nepal, there are few reports on the molecular and phylogenetic analysis of this virus. In this study, we report the circulation of seropositive occult hepatitis as well as CD-recombinant genotype in Nepalese population.

A case of high altitude cutaneous leishmaniasis in a non-endemic region in Nepal.

A case of cutaneous leishmaniasis was discovered in a 32-year old man with a persistent erythematous plaque. The patient resides in a high altitude (~2000 m above sea level) area that is not endemic for cutaneous leishmaniasis in the Dunai village of Dolpa, Nepal. The patient's lesion was initially misdiagnosed as lupus vulgaris. After response failure to initial treatment, additional testing by histological microscopy revealed the presence of Leishmania amastigotes in tissue from the lesion, and the diagnosis of cutaneous leishmaniasis was confirmed by nested PCR DNA assay of tissue from the lesion, and by a positive rK39 test in blood. Sequencing of the kinetoplast region confirmed the presence of Leishmania donovani complex. The patient responded well to treatments for cutaneous leishmaniasis and the skin lesions regressed after 6 months. This is the first known case of cutaneous leishmaniasis in a patient in Nepal who resides at high altitude in a non-endemic region. Increasing temperatures in this region of Nepal may be expanding the range of vectors that transmit cutaneous leishmaniasis.

First report of malaria parasites in water buffalo in Nepal.

We present the first molecular-based report on ungulate malaria parasites from water buffalo in Nepal. Fifty-six blood samples were collected from different groups of water buffalo (wild, feral, and domestic) and PCR assays were conducted using Plasmodium spp. cytb specific primers. Two positive cases were detected, one each from feral and domestic individuals. Complete mitochondrial genome sequence (5987 bp) was obtained and examined for nucleotide variations. Sequence analysis revealed identity with type II water buffalo malaria parasites, reported previously, with one A to T nucleotide difference at position 5344. Prevalence, as well as possible economic impacts of water buffalo malaria, should be determined on a wider set of samples from buffalo across Nepal.

Miltefosine Rescue Treatment for Visceral Leishmaniasis Relapse Patient.

We report the first case of miltefosine rescue treatment carried out for visceral leishmaniasis (VL) relapse patient. Despite undergoing treatment regimens of liposomal amphotericin B (LAMB) 5 mg/kg (standard: 10 mg/kg) daily for 3 days during the first episode followed by LAMB 5 mg/kg stat and paromomycin 15 mg/kg IM for 10 days against the second episode, the patient suffered from a third relapse and was eventually treated with miltefosine 50 mg BID directly observed treatment for 28 days. Prior to treatment, the patient had a history of intermittent fever and vague abdominal pain for one week and epistaxis for 4 days. He had massive splenomegaly, tested positive for the rK39 test, and showed LD bodies in bone marrow aspirate. The patient tested negative for malaria and HIV. Upon treatment completion with miltefosine, the patient had clinically improved and showed no LD bodies in bone marrow.

Molecular evidence supports the expansion of visceral leishmaniasis towards non-program districts of Nepal.

BACKGROUND: Visceral Leishmaniasis (VL) is caused by a protozoan parasite Leishmania donovani that is transmitted to humans by an infected female sandfly, Phlebotomus argentipes. VL is common in the Indian sub-continent including Nepal and efforts for its elimination are ongoing. However, expansion of disease towards the higher altitude areas, previously considered as VL free in Nepal, may impact the ability to achieve the elimination target by 2020. METHODS: This was an exploratory study, where VL suspected patients living exclusively in the non-program districts of Nepal and presenting with fever > 2 weeks and splenomegaly was included. The patients' blood samples were collected, and DNA was extracted. DNA was subjected to PCR amplification and subsequent sequencing. Additionally, past 10 years data of VL cases from the national databases were analysed to see the trends of the disease in program and non program districts. RESULTS: Analysis of the past 10 years data revealed that trend of VL cases significantly decreased in the program districts (p = 0.001) while it increased in the non-program districts (p = 0.002). The national trend for overall incidence of VL also significantly decreased over this time period. Limited number of patients' samples (n = 14) were subjected to molecular investigation, and four patients were found to be positive for Leishmania species by PCR. Interestingly, these cases in non-program districts were indeed also L. donovoni complex. All four patients were male with age ranges from 10 to 68 years. GenBank BLAST of the obtained DNA sequences confirmed identified specimens as L. donovani complex. We identified additional VL cases from non-program districts (including the high lands) of Nepal, indicating that the infection could be an emerging threat for the non-program areas of Nepal. CONCLUSION: The demonstration of VL cases in areas initially considered non-endemic has raised concern about on-going transmission in those regions and may trigger subsequent government plan and action to include those areas in the elimination program. Thus, the government should consider revising the disease control programs to accommodate non-program districts for achieving the VL elimination goal set for 2020.

Molecular Confirmation of G1138A Mutation in FGFR gene in Achondroplasia.

INTRODUCTION: Achondroplasia is the most common form of skeletal dysplasia of genetic origin in humans which is characterized by disproportionate rhizomelic dwarfism. Heterozygous mutation in the transmembrane domain of the FGFR3 gene (4p16.3) occurs as a de novo mutation in most of the cases. METHODS: DNA was isolated from seven samples, out of which, five had clinical features of Achondroplasia while one was dwarf but did not show symptoms of the disorder and one as negative control. PCR was performed for the region incorporating the hotspot region viz. 1138th nucleotide. PCR amplicon of size 164 bp was obtained from all the samples, and was sequenced. RESULTS: Sequence analysis showed the presence of mutation (G to A transition) in all of the five samples. The five samples that showed the clinical features of Achondroplasia had mutation in the region being analyzed while the single patient who had no clinical manifestations of the disorder despite being dwarf had no such mutation. Among the five patients studied, one patient had a family history of Achondroplasia as observed through pedigree analysis while the remaining four cases were sporadic in nature. CONCLUSIONS: This study further supports that the G1138A mutation is the one of the most common point mutation among Achondroplasia cases. Genetic diagnosis can be useful to identify the disease prenatally and differentiate other life threatening dwarfism for the safety of both mother and fetus.

Visceral leishmaniasis from a non-endemic Himalayan region of Nepal.

Visceral leishmaniasis (VL) is endemic to the southern plains of Nepal. Here, we report the first case of VL from a non-endemic Himalayan region of Nepal. The patient presented with a history of high-grade fever, splenomegaly, and anemia but had not traveled to a VL-endemic region. Visceral leishmaniasis was diagnosed following microscopic detection of the Leishmania species amastigote in a bone marrow aspirate, positive result for the rK39 test, and further validation by nested polymerase chain reaction (PCR). The patient was treated with 5 mg/kg liposomal amphotericin B and was clinically improved upon discharge. Our result suggests that VL is expanding towards non-endemic regions of Nepal, and it should therefore be considered that VL surveillance systems be strengthened, particularly for non-program districts and VL be included as a differential diagnosis in febrile illnesses.

Investigation of antioxidant, antimicrobial and toxicity activities of lichens from high altitude regions of Nepal.

BACKGROUND: Several lichen species are reported to be used tradiationally in many theraupatic practices. Many lichen species are reported as sources of several bioactive natural compounds. Several lichen species of Nepal are so far chemically unexplored. METHODS: The morphological, anatomical and phytochemical characteristics of lichens were compared for the taxonomic identification of the species. Methanol- water extract of lichens were sub fractionated into hexane, dichloromethane and methanol fractions for bioactivity assays. Antimicrobial activities of extracts were evaluated agaisnt pathogenic bacteria and fungal species. DPPH test was used for antioxidant potential evaluation. Brineshrimp test was perfermed to evaluate toxicity of the extracts. RESULTS: A total of 84 lichen specimens were collected and identified from Annapurna Conservation Area (ACA) Nepal. The specimens were identified as belonging to 19 genera and 47 species. Methanol fractions of 16 specimens and dichloromethane (DCM) fractions of 21 lichens specimens showed antioxidant activities comparable with commercial standards (BHA, Butylated hydroxyanisole, IC50=4.9±0.9 µg/mL) even at crude extract level. Similarly, the DCM fraction of 17 lichens showed potential antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus KCTC3881) and DCM fractions of 45 lichens showed antimicrobial activity against a Gram-negative bacterium (Klebsiella pneumoniae KCTC2242). DCM fractions of three lichens showed antifungal activity against the yeast, Candida albicans KCTC 7965. Likewise, methanol fractions of 39 lichens and DCM fractions of 74 lichens showed strong toxicity against brine shrimp nauplii with more than 80% mortality. CONCLUSION: Such biological activity-rich lichen specimens warrant further research on exploration of natural products with antioxidant, antimicrobial and anti cancer (toxic) potential.