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In 2019, we surveyed the Karuna bat cave during spring and autumn seasons and recorded two species of fruit bats; Rousettus leschenaultii and Eonycteris spelaea, and two species of insectivorous bats; Hipposideros armiger and Rhinolophus macrotis. Although there was no variation in colony size of insectivorous bats, the fruit bat population (around 38,000 individuals) was only recorded in autumn which indicated seasonal migration. Occasional killing of bats, unsustainable guano extraction, and tourism development threaten this major cave for bats in Nepal. The study recommends prioritizing scientific research and bat friendly cave conservation actions.

Latrine sites are the places used for urination and defecation, which mostly act as a signaling agent for multiple purposes like territorial marking, confrontation with extruders or potential predators, delivering different inter and intra-communication messages. To understand latrine site visit pattern, a single camera trap was deployed for 91 trap nights at the latrine site of Large Indian Civet during the months of December 2016 and February & March 2017. Latrine site was found under the tree with abundant crown cover and bushes. At least two individuals were found to be using a single latrine site in an irregular manner between 1800 h and 0600 h with higher activity between 1800 h and 2300 h. Our results indicated an irregular latrine site visit pattern, hence similar studies with a robust research design in larger areas are required to understand specific latrine use patterns.

Background: Physiological alterations manifesting during pregnancy may influence oral health of women by increasing the susceptibility to oral infections. This study was carried out to assess oral hygiene status, gingival status, periodontal status and treatment needs (TNs) among pregnant and nonpregnant women. Methods: A comparative cross-sectional study was conducted in patients attending Outpatient Depart­ment of Obstetrics and Gynaecology, Chitwan Medical College and Teaching Hospital, Bharatpur, Chit­wan, Nepal from December 2020 to June 2021. A total of 102 pregnant (34 in each trimester) and 102 nonpregnant women of 15-49 years fulfilling all inclusion criteria were evaluated for oral health status by various indices including oral hygiene index-simplified (OHI-S), gingival index (GI), and community periodontal index and treatment needs (CPITN). Convenience sampling technique was done. Data was analysed in SPSS v.26.0. Results: The mean scores of OHI-S, GI and CPITN were statistically (p value

Background: Breech presentation has been associated with higher rates of perinatal morbidity irrespective of mode of delivery. The aim of this study was to determine the prevalence and perinatal outcomes of singleton breech deliveries at Chitwan Medical College, a tertiary level hospital in Nepal. Methods: A retrospective review of records of all women who delivered at Chitwan Medical College with breech presentation from September 2018 to October 2020 was conducted. Information on demographic variables, obstetric characteristics and perinatal outcomes were obtained, recorded and analyzed. Results: Out of 6712 cases of deliveries during the study period, 226 had breech presentation with prevalence of 3.37%. One hundred ninety-one (84.5%) of them had undergone Caesarean delivery with 125 (65.4%) emergency and 66 (34.6%) elective Caesarean delivery. More than two third (70.4%) of breech deliveries were term deliveries while 28.8% were preterm. There were 2 cases of still births and 1 neonatal death with Perinatal Mortality Rate of 13.27 per 1000 breech deliveries. Vaginal breech delivery was associated with higher perinatal mortality, low Apgar score and low birth weight. Caesarean breech delivery was associated with increased rates of Neonatal Intensive Care Unit admission. Conclusions: The neonatal mortality and morbidity were higher with the vaginal breech deliveries. However further studies with larger sample size and greater statistical power is necessary to draw definitive conclusion.

In this paper, an attempt has been made to develop a Python module for evaluating energy in transit (viz., heat and work). The work considered in this article is non-dissipative displacement work. Functions for constant pressure, volume, and temperature were developed, along with adiabatic and polytropic processes. Also, the sensible and latent heat were also transformed into functions. The modules NumPy and Matplotlib were used to perform the stipulated task. The correctness of codes was checked against four different numerical problems, and it has been observed that the program results match exactly with the results in the literature. As a result, the developed functions thus developed have shown high accuracy with the least effort and error in all the cases

Out of the four Molossidae species from South Asia, the distribution of the European free-tailed bat, T. teniotis is most poorly known. This species has been occasionally reported from Afghanistan, Bangladesh, Bhutan, and India; however, no records exist in Nepal. Here we report the first record of T. teniotis from Nepal and comment on its possible elevational movement in the Himalaya. Active acoustic surveys were conducted in the Kali Gandaki canyon during autumn and winter seasons at two elevational zones, 800–1200 m and 2100–2500 m, in three habitat types (forest, agricultural land, and human settlements). Echolocation calls of T. teniotis were easily distinguished by their low frequency, shallow frequency modulation, and long duration. During autumn, the activity was recorded only at 2100 to 2500 m and varied significantly from winter activity, while T. teniotis was observed at both elevational zones during winter. The result confirms the presence of T. teniotis from Nepal. Based on our observations of differential activity at different elevation zones in two seasons, we recommend more intensive studies to confirm seasonal migration and to understand seasonal demographics along the Kali Gandaki landscape and in the entire Himalayan range at large.

Bat research in Nepal is limited and most areas remain to be explored. Sparse research has been conducted in the Banpale forest and to improve the understanding of bat diversity, an updated species checklist was prepared. Trapping surveys using mist nets were conducted at four different locations in the forest from March to May 2018. This survey identified 55 individuals belonging to eight species within eight genera and four families. Trapping locations near less disturbed forest edges and water resources were found to have higher bat diversity compared to highly disturbed areas (e.g., landslides and logging areas). With information from survey and secondary source, we conclude that Banpale forest harbours 10 of the 53 bat species found in Nepal. We recommend adopting practices to conserve the forest from landslides and minimizing illegal logging to conserve bat forest habitats.

Background: Hysterectomy is an effective treatment for a wide range of gynaecological diseases, both benign and malignant. There must be a pe­riodic audit for the appropriate indication of the surgery and its complica­tion rate. Histopathological analysis is mandatory for definitive diagnosis and further management. This study aims to review the clinical profile, indications of hysterectomy and to assess the correlation between the clinical diagnosis and histopathological report. Methods: A retrospective study of all the patients undergoing abdominal hysterectomy was conducted in the Department of Obstetrics and Gyne­cology, Chitwan Medical College Teaching Hospital, Nepal from January to December 2018. Demographic and clinical informations were retrieved from the medical record section. Datas were entered in the pre-designed proforma. Statistical analysis was done in terms of percentages, standard deviation, correlation and mean. Results: During the study period, 111 patients underwent abdominal hys­terectomy. The mean age of the patients was 46 years. The mean dura­tion of hospital stay was 8.7 days. Abnormal menstrual flow and pain ab­domen were the commonest presenting symptoms. The most common preoperative diagnosis was fibroid followed by ovarian tumor. Leiomyoma was the commonest lesion on histopathological examination. The correla­tion between clinical diagnosis and histopathology was 96.07% for fibroid, 80.95% for ovarian tumor, 66.67% for adenomyosis and 38.46% for DUB. Conclusions: Fibroid uterus and ovarian mass are the common indications for abdominal hysterectomy. Histopathology is mandatory for confirming the diagnosis, proper counseling and holistic management of the patient.

Background: Cervical cancer ranks the most common cancer among Nepalese women. Cervical and vaginal infections constitute the most common gynecological problems. Through Pap smear screening, early diagnosis, follow up and treatment, morbidity and mortality can be reduced by 70% and 80% respectively. Methods: This retrospective study was conducted to know the incidence of abnormal cervical epithelial lesions and cervico-vaginal infections. It was carried out in the Outpatient Department of Gynecology at Chitwan Medical College from April 2018 to March 2019. The data were collected and standard statistical analysis done using Microsoft Excel 2007. Results: The most prevalent cervical epithelial cell abnormality was atypical squamous cells of undetermined significance (3.43%) whereas the least common was low grade squamous intraepithelial lesion (0.21%). The most prevalent cervical vaginal infection was Bacterial vaginosis (12.30%) whereas the least prevalent was Trichomoniasis (0.29%). The majority of the women with epithelial cell abnormality were in the age group of 31-40 years whereas the lowest number in the age group less than 20 years. The highest prevalence of cervical vaginal infection was in the reproductive age group (31-50 years) and the lowest in the age above 51 years. Most of the patients had multiple symptoms like vaginal discharge, genital itching, whereas only (20.17%) visited for routine screening. Conclusion: Atypical squamous cells of undetermined significance and Bacterial vaginosis were the most common finding in Pap smear test representing abnormal cytology of cervix and cervicovaginal infection respectively.

This study provides an insight into the comparison of the tensile properties between 17-4 PH SS samples processed by Selective Laser Melting (SLM) and the wrought 17-4 PH samples. The tests were conducted according to the international standard ASTM E8 and the microstructural studies were also performed. The SLM built part revealed superior tensile properties against the wrought sample. Furthermore, the SLM processed sample displayed significantly higher ductility and ultimate tensile strength (UTS) with respect to wrought samples. The microstructure of the SLM-built samples showed higher proportion of retained austenite compared to wrought samples.

Miscarriage is a frequent outcome of pregnancy, with major emotional implications to the couple experiencing such an event. Threatened miscarriage is the commonest complication of early pregnancy and affects about 20% of pregnancies. It presents with vaginal bleeding with or without abdominal cramps. On the other hand recurrent miscarriages are post implantation failures in natural conception. Increasing age of women, smoking, obesity or polycystic ovary syndrome (PCOS) and a previous history of miscarriage are risk factors for threatened miscarriage. The pathophysiology has been associated with changes in levels of cytokines or maternal immune dysfunction. Clinical history and examination, maternal serum biochemistry and ultrasound findings are important to determine the treatment options and provide valuable information for the prognosis. Many surgical and non-surgical interventions are used in the management of threatened and recurrent miscarriages. In this review, we present available evidence-based guidance on the incidence, pathophysiology, investigation and clinical management of recurrent miscarriage and threatened miscarriage, focusing mainly on the first trimester of pregnancy and primary healthcare settings. The review is structured to be clinically relevant. We have critically appraised the evidence to produce a concise answer for clinical practice.

Background: There is no general consensus about the role of routine HPE of the obtained tissue at the time of uterine evacuation. However, it is understood clinically that it is of utmost importance to prove the presence of intrauterine gestation and to exclude gestational trophoblastic disease in the form of partial or complete mole. This study aimed to assess the role of histopathology in cases of first trimester miscarriages and to determine clinical relevance of histopathological examination following surgical evacuation. Methods: This was a retrospective study of collected data over 12 months (January 2018- De­cember 2018) in an agency for reproductive health setup with predefined inclusion criteria. We included 60 consecutive patients attended with history of first trimester bleeding. Patient’s record and the histopathological examination report of products of conception following uterine evacu­ation were observed. The data was analyzed descriptive statistics including percentage, stander deviation, mean, and range in Microsoft Excel software. Results: Missed abortion was the most common type and constituted 55% of the studied group. The histopathological reports confirmed the pregnancy in all patients and revealed partial mole in 6(10%) patients and complete mole in 2(3.33%) patients. Conclusions: Histopathological examination of products of conception detects under diagnosed molar pregnancies that necessitates special follow up and is a key step to do further management.

Background: Labor induction is one of the most common Obstetrics interventions worldwide. It has got significant risks and benefits. Careful selection of cases for induction improves the feto­maternal outcomes. So, the study was designed to assess the fetomaternal outcome and factors associated with mode of delivery following the induction of labor. Methods: It was a descriptive observational study conducted in the department of Obstetrics and Gynecology from July 17, 2018, to July 16, 2019. The study comprised of 264 mothers admitted in the labor ward subjected to labor induction as clinical management of labor and delivery. Data analyzed with chi-square test and one-way analysis of variance (ANOVA) wherever appropriate. Results: Of 2950 deliveries, the induction rate was 8.94%. Out of them, 264 mothers undergone labor induction. A caesarean was the highly associated mode of deliveries 168(63.63%) due to in­duction failure 94(55.97%) followed by fetal distress 37(22%). The mean birth weight of neonates was 3.09±0.41 kg. Apgar score in one minute and hospital stay showed a significant difference (p-value .002), in modes of deliveries. No significant association was observed in maternal and fetal complications like PPH, endometritis, and low Apgar score

COVID-19 is a pandemic affecting every group of people. Pregnancy itself is a partially immune compromised state, so chance of aggravation of symptom supposed to be more in pregnancy. Prevention is the best way to get away from this disease. Good hygiene, supportive care and possible mechanical ventilation for severe case are required. There is no proven role of antibiotic, antiretroviral and steroids. There is no proven vertical transmission. Baby has to be kept in isolation and breast milk can be expressed and given to newborn. Keywords: COVID-19, pandemic, pregnancy.

A bamboo bat of the genus Tylonycteris was captured near Gupteshore Cave of Kushma in Parbat, Nepal. Traditionally, two species of Tylonycteris (of T. pachypus complex and T. robustula complex) are known from the Indian subcontinent. Due to inconsistency in taxonomic classification, several changes were recently made within the genus Tylonycteris—T. pachypus was corrected to T. fulvida and T. robustula to T. malayana. The occurrence of Tylonycteris from Nepal’s diversified zoogeography, however, was never mentioned. This note provides a new record of Tylonycteris from Nepal. Based on morphological characteristics and species distribution range, this note confirms the captured species as T. fulvida.

DOI: https://doi.org/10.3126/jcmc.v9i1.23779 Background: Emergency Peripartum Hysterectomy (EPH) is an important lifesaving surgical procedure considered in cases of severe hemorrhage unresponsive to medical and conservative management. The objective is to review incidence, identification, intervention and impact of emergency peripartum hysterectomy. Methods: The retrospective, cross-sectional study designed was to used. EPH data were collected from January 2014 to December 2018.Descriptive statistics was used to analyzed data and presented in tables and charts. Results: Incidence of Emergency Peripartum Hysterectomies was 2.3% out of 252(2.6%) cases of obstetrical emergencies and 0.06% that is 1 in 1600 deliveries. Most common indications for EPH were uterine rupture (33.3%); placenta accreta (33.3%) followed by retained placenta (16.6%) and endometritis with pyometritis (16.6%). Estimated blood loss 1916 ml., timeliness from delivery to hysterectomy was 140 minutes; most common post-operative complication was surgical site infection (33.3%) and length of hospital stay 11.7 days. Maternal morbidity rate was 33.3%. There was no maternal mortality recorded. Conclusions: The timely intervention improves the outcome in Peripar­tum Hysterectomy, which is frequently associated with abnormal placen­tation as a consequence of increasing caesarean deliveries rate. Keywords: Cesarean hysterectomy, Peripartum hyterectomy, Postpartum hysterectomy

Despite reports of pulmonary toxicity due to styrene, guidelines on acceptable styrene exposure levels have been based on risk of cancer and central nervous system and liver toxicity and not on respiratory effects. Many reports have linked exposure to styrene vapor in occupational settings to various forms of non-malignant pulmonary disorders including bronchiolitis, hypersensitivity pneumonitis, and occupational asthma. We report two cases in which the same tasks performed in a single workplace resulted in exposure to styrene vapor with subsequent development of acute respiratory symptoms associated with impaired gas exchange and imaging and histopathologic findings consistent with bronchiolitis and organizing pneumonia. Both patients gradually recovered once their workplace exposure to styrene was terminated. Clinicians, employers, and insurers should be aware of the potential for pulmonary toxicity from exposure to styrene.

ObjectivesStillbirth is one of the vital indicators of quality care. This study aimed to determine maternal-fetal characteristics and causes of stillbirth in Nepal.DesignSecondary analysis of single-centred registry-based surveillance data.SettingThe study was conducted at the Department of Obstetrics and Gynecology, Chitwan Medical College Teaching Hospital, a tertiary care hospital located in Bharatpur, Nepal.ParticipantsAll deliveries of intrauterine fetal death, at or beyond 22 weeks’ period of gestation and/or birth weight of 500 g or more, conducted between 16 July 2017 and 15 July 2019 were included in the study.Main outcome measuresThe primary outcome measure of this study was stillbirth, and the secondary outcome measures were maternal and fetal characteristics and cause of stillbirth.ResultsOut of 5282 institutional deliveries conducted over 2 years, 79 (1.5%) were stillbirths, which gives the stillbirth rate of 15 per 1000 births. Of them, the majority (75; 94.9%) were vaginal delivery and only four (5.1%) were caesarean section (pConclusionHigh rate of stillbirths in Nepal could be due to the lack of quality antenatal care. The country’s health systems should be strengthened so that pregnancy-related risks such as maternal hypertension and infections are identified early on. Upgrading mothers’ hygiene and health awareness is equally crucial in reducing fetal deaths in low-resource settings.

Bovine enzootic haematuria (BEH) is a debilitating disease of cattle caused by chronic ingestion of bracken fern. Control of BEH is difficult when bracken fern is abundant and fodder resources are limited. To fill a significant knowledge gap on modifiable risk factors for BEH, we conducted a case-control study to identify cattle management practices associated with BEH in the Bhutanese cattle population. A case-control study involving 16 of the 20 districts of Bhutan was carried out between March 2012 and June 2014. In Bhutan sodium acid phosphate and hexamine (SAP&H) is used to treat BEH-affected cattle. All cattle greater than three years of age and treated with SAP&H in 2011 were identified from treatment records held by animal health offices. Households with at least one SAP&H-treated cattle were defined as probable cases. Probable case households were visited and re-classified as confirmed case households if the BEH status of cattle was confirmed following clinical examination and urinalysis. Two control households were selected from the same village as the case household. Households were eligible to be controls if: (1) householders reported that none of their cattle had shown red urine during the previous five years, and (2) haematuria was absent in a randomly selected animal from the herd following clinical examination. Details of cattle management practices were elicited from case and control householders using a questionnaire. A conditional logistic regression model was used to quantify the association between exposures of interest and household BEH status. A total of 183 cases and 345 controls were eligible for analysis. After adjusting for known confounders, the odds of free-grazing for two and three months in the spring were 3.81 (95% CI 1.27-11.7) and 2.28 (95% CI 1.15-4.53) times greater, respectively, in case households compared to controls. The odds of using fresh fern and dry fern as bedding in the warmer months were 2.05 (95% CI 1.03-4.10) and 2.08 (95% CI 0.88-4.90) times greater, respectively, in cases compared to controls. This study identified two husbandry practices that could be modified to reduce the risk of BEH in Bhutanese cattle. Avoiding the use of bracken fern as bedding is desirable, however, if fern is the only available material, it should be harvested during the colder months of the year. Improving access to alternative fodder crops will reduce the need for householders to rely on free-grazing as the main source of metabolisable energy for cattle during the spring.

An investigation was carried out to evaluate the susceptibility of rice cultivation to major field diseases like brown spot, sheath blight and bacterial leaf blight under middle Indo Gangetic Plain. The disease incidence and disease severity were observed at three different growth stages namely flowering, milk and maturity in boro seasons during 2015-16. The incidence of brown spot ranged from 8.93 to 17.83%, 16.67 to 25.67% and 18.33 to 28.33% at flowering, milk and maturity stages, respectively. The severity of brown spot in grade (0-9 scale) ranged from 5.13 to 10.33, 9.33 to 20.00 and 11.57 to 22.67, respectively at flowering, milk and maturity stages. The incidence of sheath blight of paddy ranged from 7. 03 to 16.50%, 13.13 to 22.67% and 19.67 to 35.67% at flowering, milk and maturity stages, respectively. The severity of sheath blight in grade (0-9 scale) ranged from 4.00 to 8.60, 8.33 to 13.93 and 18.33 to 34.33, respectively at flowering, milk and maturity stages. The incidence of bacterial leaf blight ranged from 5.00 to 14.00%, 11.43 to 21.00% and 17.00 to 34.00% at flowering, milk and maturity stages, respectively. The severity of bacterial leaf blight in grade (0-9 scale) ranged from 2.00 to 8.00%, 6.33 to 12.05% and 14.47 to 20.17%, respectively at flowering, milk and maturity stages. Among the varieties, the highest incidence and severity of Brown spot was recorded on Rajendra Bhagawati whereas it was lowest on 27P31 at all growth stages. In the case of Sheath blight, highest incidence and severity was recorded on Sambha Mahsuri whereas it was the lowest on CRL 193. Beside this in the case of Bacterial leaf blight, the highest incidence and severity was recorded on Kranti whereas the lowest was recorded on CRL 193. In general, it was observed that the incidence and severity of diseases increased gradually from flowering to maturity stage and the genotypes with the minimum incidence and severity of diseases gave the maximum yield.

Balloon Mitral Valvotomy (BMV) is one the preferred modality of treatment for Rheumatic mitral stenosis. This is usually performed with onsite surgical backup to tackle the complications like cardiac tamponade needing surgical drainage and severe mitral regurgitation which requires emergency Mitral Valve Replacement. Our aim is to evaluate the pre and post procedural outcomes of the BMV, without onsite surgical backup. Total patients were 88 who underwent BMV in Chitwan Medical College, from Feb 2014 to Jan. 2016. All symptomatic patients (NYHA class II- IV) with their mitral valve area (MVA )

Rabies remains a disease of significant zoonotic and economic concern in rabies endemic areas of Bhutan. Rabies outbreaks in livestock threaten the livelihoods of subsistence farming communities and pose a potential public health threat. As a part of identifying approaches to prevent rabies in cattle, a Knowledge, Attitude, and Practice (KAP) survey was conducted among cattle owners in selected rural areas of the southern rabies high-risk zone and low-risk zone in eastern Bhutan. Between March and April 2017, 562 cattle owners (281 in the east and 281 in the south) were interviewed using a questionnaire. Eighty-eight percent of the participants had heard of rabies but only 39% of the participants who had heard of rabies had adequate knowledge about rabies. Multivariable logistic regression analysis showed that residing in the south [OR = 9.25 (95% CI: 6.01-14.53)] and having seen a rabies case [OR = 2.46 (95% CI: 1.6-3.82)] were significantly associated with having adequate knowledge about rabies. Based on our scoring criteria, 65% of the total participants who had heard of rabies had a favorable attitude towards rabies control and prevention programs. The participants residing in the east were two times more likely to have a favourable attitude than their counterparts in the south [OR = 2.08 (95% CI: 1.43-3.05)]. More than 70% of the participants reported engaging in farm activities such as examining the oral cavity of sick cattle and assisting cattle during parturition. Only 25% of the participants reported using personal protective equipment while undertaking these activities. Despite a high level of rabies awareness, we observed that there is a lack of comprehensive knowledge about rabies regarding susceptible hosts, transmission routes, the health outcome of rabies infection in humans, and appropriate health-seeking behaviours. This study highlights the need to strengthen rabies education programs in rural communities to address the knowledge gaps that have been identified.

The present investigation was undertaken to study the effect of silymarin on cardiac hypertrophy induced by partial abdominal aortic constriction (PAAC) in Wistar rats. Silymarin was administered for 9 weeks at the end of which we evaluated hypertrophic, hemodynamic, non-specific cardiac markers, oxidative stress parameters, and determined mitochondrial DNA concentration. Hypertrophic control animals exhibited cardiac hypertrophy, altered hemodynamics, oxidative stress, and decreased mitochondrial DNA (mtDNA) concentration. Treatment with silymarin prevented cardiac hypertrophy, improved hemodynamic functions, prevented oxidative stress and increased mitochondrial DNA concentration. Docking studies revealed that silymarin produces maximum docking score with mitogen-activated protein kinases (MAPK) p38 as compared to other relevant proteins docked. Moreover, PAAC-control rats exhibited significantly increased expression of MAPK p38β mRNA levels which were significantly decreased by the treatment of silymarin. Our data suggest that silymarin produces beneficial effects on cardiac hypertrophy which are likely to be mediated through inhibition of MAPK p38β.

Study of mammals outside protected area is limited in Nepal especially small patches of forest used by small carnivores and very less is known about current biodiversity status. With the importance of research in such area, this study was carried out in the Banpale forest using non-invasive method camera trapping and past records as a tool to monitor the mammals. A total of 20 mammal species were recorded in 36.33 ha patch of forest in between December, 2016 to March, 2017. Large Indian civet has been trapped in the camera for the most time during the entire survey. Common leopard was also sighted and camera trapped however the prey species were neither camera trapped nor any direct sighting was made. The forest has a wide diversity of least researched small mammals which have been trapped by the camera. The threats have been in the peak causing negative interaction between the wildlife and humans. Conservation action plan for the wildlife should be duly implemented with strict rules and regulations for the conservation of remaining wildlife present in the forest.

TOC summary: A major outbreak affected dogs, domestic livestock, and humans., From January through July 2008, rabies reemerged in the Chhukha district of southwestern Bhutan. To clarify the distribution and direction of spread of this outbreak, we mapped reported cases and conducted directional tests (mean center and standard deviational ellipse). The outbreak resulted in the death of 97 animals (42 cattle, 52 dogs, and 3 horses). Antirabies vaccine was given free of charge to ≈674 persons suspected to have been exposed. The outbreak spread south to north and appeared to follow road networks, towns, and areas of high human density associated with a large, free-roaming, dog population. The outbreak was controlled by culling free-roaming dogs. To prevent spread into the interior of Bhutan, a well-coordinated national rabies control program should be implemented in disease-endemic areas.

Immunogenicity as a potential consequence of therapeutic protein administration is increasingly being scrutinized in the biopharmaceuticals industry, particularly with the imminent introduction of biosimilar products. Immunogenicity is an important safety aspect requiring rigorous investigation to fully appreciate its impact. Factors involved in product handling, such as storage temperature, light exposure, and shaking, have been implicated in immunogenicity, while container closure systems are no less important. Intended to provide a stable environment for the dosage form, container closures may also interact with a product, affecting performance and potentially enhancing immunogenicity. Glass surfaces, air-liquid interfaces, and lubricants can mediate protein denaturation, while phthalates in plastics and latex rubber are sources of extractables and leachates that may contaminate a product, causing allergic reactions and increasing immunogenicity. The manufacture of therapeutic proteins therefore requires rigorous safety evaluations not just in the context of the product, but also product containment.

The patents of first-generation biopharmaceutical proteins are expiring, creating opportunities for biosimilar products. Unlike conventional generic pharmaceuticals, the development of biosimilar products is far more complex and requires more than a simple demonstration of pharmacological bioequivalence to establish efficacy and safety. The main concern with biosimilar products, as for any therapeutic protein, is immunogenicity and with it the potential for serious clinical sequelae. In the absence of adequate predictors of immunogenicity outside the clinical trial setting, biosimilar products should be evaluated in the same way that any novel pharmaceutical is evaluated. Herein, the factors involved in breaking host tolerance following administration of a therapeutic protein are discussed. The impact of product handling on immunogenicity is considered in the context of some hard-fought lessons that have helped to shape the current era of biopharmaceutical manufacturing, packaging, distribution, storage, and quality assurance.

Human erythropoietin produced by recombinant DNA technology, is now marketed worldwide for the treatment of anemias associated with chronic renal failure and chemotherapy. No sensitive methods, which can determine r-HuEPO dimer or oligomer aggregate content in formulated products, have been published to date. This report describes the development and validation of a sensitive size exclusion high performance liquid chromatography (HPLC) method for the quantitation of r-HuEPO aggregates in formulations containing 0.03% polysorbate 80. A Waters Alliance 2690 HPLC system connected to a TosoHaas TSKgel G3000 SWxl (7.8 mm x 30 cm, 250 A pore size, 5 microm particle size) column and a Waters 474 fluorescence detector was used. The mobile phase for the SEC-HPLC method consists of isopropyl alcohol-potassium phosphate (0.1 M)/potassium chloride buffer (pH 6.8+/-0.1, 0.2 M) (25:75, v/v). The flow rate was 0.3 mL/min and the method run time was 60 min. The SEC-HPLC method presented here was shown to be specific for r-HuEPO total aggregates (dimer and oligomers) and allows for their quantitation at 80 ng/mL or 4 ngs/injection, in the presence of r-HuEPO monomer and the pharmaceutical excipients, glycine (5 mg/mL), sodium chloride (4.3 mg/mL), and 0.03% polysorbate 80. The finalized method is stability-indicating and is suitable for determining r-HuEPO aggregates between 0.2 and 0.5% levels in the formulated product of r-HuEPO. This method offers a robust way to measure total aggregates on a routine basis with a high sensitivity for use in product quality control.

We describe the population demographics, rudimentary measures of reproductive performance, the prevalence of major disease conditions and reason-specific proportional mortality for cattle owned by villagers in 16 dzongkhags (districts) in Bhutan based on the findings of a cross-sectional study carried out between March 2012 and May 2014. The animal health issues that were of concern for livestock owners are also identified. Study households were selected using a stratified, two-stage cluster design. Districts (dzongkhags) formed the strata. Villages within dzongkhags were the first sampling stage and households within villages the second sampling stage. All cattle within each selected household comprised the study population. Questionnaires were collected from 409 households with 1480 standing cattle. A total of 71.0% (95% CI 67.9%-74.1%) of the standing cattle population were female and the median age of cattle was 5 years (Q25 3 years; Q75 7 years). Exotic breeds of cattle (Jerseys, Brown Swiss, and their crosses) comprised 41.4% of the cattle population with local breeds making up the remainder. Although exotic breeds of cattle had a significantly lower age at first calving (median 4 years) compared to local breeds (median 4.8 years) there was no significant difference in the number of calving events per cow for the two breed groups. Diarrhoea was the most prevalent disease condition with 2.8 (95% CI 1.5-4.6) cases per 100 animals followed by bovine enzootic haematuria with 1.9 (95% CI 1.0-3.3) cases per 100 animals. The most frequently cited cause of death was misadventure (proportional mortality 26.2%, 95% CI 15.7%-39.2%) followed by old age (17.8%, 95% CI 9.5%-29.4%). A lack of access to adequate fodder and pasture was the animal health issue that was cited by interviewees most frequently. We provide no evidence that exotic breeds of cattle have superior reproductive performance compared with local breeds. The major cattle health concern cited by interviewees, lack of access to fodder, is likely to contribute to suboptimal productivity and the relatively high incidence of bovine enzootic haematuria in this population. We propose that a prospective cohort study of Bhutanese cattle is warranted to more fully elucidate factors affecting the productivity and longevity of cattle in Bhutan.

The incidence of pure red cell aplasia (PRCA) in patients with chronic kidney disease associated with the subcutaneous (s.c.) administration of epoetin alfa (EPREX®) began to increase in 1998. As part of an intensive investigation into the reasons for this increase, in vivo models were developed to assess the ability of potential causative factors to stimulate an immune response to recombinant human erythropoietin (rHuEPO). It was difficult to generate anti-EPO antibodies in mice. In animals injected with rHuEPO alone, anti-EPO antibodies were either absent or present at very low levels. The addition of an adjuvant to the immunization protocol was able to increase both the frequency of occurrence and titer of the immune response and resulted in the generation of anti-EPO antibodies that, in most cases, recognized both human and mouse EPO. Some mice exhibited a reduction in hematocrit, suggesting neutralization of endogenous EPO by anti-EPO antibodies. To evaluate the primary lead identified in the technical investigation, leachates from the uncoated syringe stoppers of EPREX® syringes, a surrogate antigen (chicken egg albumin, OVA) was used to avoid possible interferences that could arise from the use of an endogenous protein like EPO. These leachates yielded a positive, concentration-dependent antibody response in the OVA animal model, demonstrating their adjuvant properties and providing support for the hypothesis generated through the technical investigation that leachates were capable of enhancing the immune response to rHuEPO.

The phases formed by 10, 20, 30, 40 and 50 mg ml-1 solutions of a compound, named RWJ-47428, in water have been studied with optical polarizing microscopy and X-ray diffraction. All solutions were found to exhibit the nematic liquid crystalline phase over a wide temperature range. Changes in optical textures showed a transition to the isotropic phase above 130°C in the most concentrated solution. The dimensions (length and width) of the orienting unit were determined to be 22 A and 4.9 A. Higher concentration samples exhibit both the nematic and the lamellar phases.

The stability, potency, and preservative effectiveness of two dilutions of epoetin alfa containing a bacteriostatic diluent were studied. Epoetin alfa 10,000 units/mL in single-use vials was diluted 1:1 and 1:1.5 with bacteriostatic 0.9% sodium chloride injection. USP tests of preservative effectiveness were performed on samples from two batches each of the 1:1 and the 1:1.5 dilutions. Appearance assessment, Western blot analysis, radioimmunoassay, and bioassay were used to determine the stability or potency of samples from three batches of each dilution that were stored at 5 degrees C or at 30 degrees C for 12 weeks. Both batches of the 1:1.5 dilution (epoetin alfa 4,000 units/mL with 0.54% benzyl alcohol) met the USP criteria for preserved solutions, while one batch of the 1:1 dilution (epoetin alfa 5,000 units/mL with 0.45% benzyl alcohol) did not. Epoetin alfa 10,000 units/mL diluted either 1:1 or 1:1.5 with bacteriostatic 0.9% sodium chloride injection and stored at 5 degrees C or at 30 degrees C remained stable and potent for 12 weeks. The addition of 1.5 mL of bacteriostatic 0.9% sodium chloride injection to a vial containing 1 mL of epoetin alfa 10,000 units/mL makes a solution of epoetin alfa 4,000 units/mL that meets the USP criteria for preservative effectiveness and remains stable and potent for 12 weeks at 5 degrees C.