Estimating the Seroincidence of Scrub Typhus using Antibody Dynamics after Infection.

Scrub typhus, a vector-borne bacterial infection, is an important but neglected disease globally. Accurately characterizing the burden is challenging because of nonspecific symptoms and limited diagnostics. Prior seroepidemiology studies have struggled to find consensus cutoffs that permit comparisons of estimates across contexts and time. In this study, we present a novel approach that does not require a cutoff and instead uses information about antibody kinetics after infection to estimate seroincidence. We use data from three cohorts of scrub typhus patients in Chiang Rai, Thailand, and Vellore, India, to characterize antibody kinetics after infection and two population serosurveys in the Kathmandu Valley, Nepal, and Tamil Nadu, India, to estimate seroincidence. The samples were tested for IgM and IgG responses to Orientia tsutsugamushi-derived recombinant 56-kDa antigen using commercial enzyme-linked immunosorbent assay kits. We used Bayesian hierarchical models to characterize antibody responses after scrub typhus infection and used the joint distributions of the peak antibody titers and decay rates to estimate population-level incidence rates in the cross-sectional serosurveys. Median responses persisted above an optical density (OD) of 1.8 for 23.6 months for IgG and an OD of 1 for 4.5 months for IgM. Among 18- to 29-year-olds, the seroincidence was 10 per 1,000 person-years (95% CI, 5-19) in Tamil Nadu, India, and 14 per 1,000 person-years (95% CI: 10-20) in the Kathmandu Valley, Nepal. When seroincidence was calculated with antibody decay ignored, the disease burden was underestimated by more than 50%. The approach can be deployed prospectively, coupled with existing serosurveys, or leverage banked samples to efficiently generate scrub typhus seroincidence estimates.

Environmental sampling for typhoidal Salmonellas in household and surface waters in Nepal identifies potential transmission pathways.

INTRODUCTION: Salmonella Typhi and Salmonella Paratyphi, fecal-oral transmitted bacterium, have temporally and geographically heterogeneous pathways of transmission. Previous work in Kathmandu, Nepal implicated stone waterspouts as a dominant transmission pathway after 77% of samples tested positive for Salmonella Typhi and 70% for Salmonella Paratyphi. Due to a falling water table, these spouts no longer provide drinking water, but typhoid fever persists, and the question of the disease's dominant pathway of transmission remains unanswered. METHODS: We used environmental surveillance to detect Salmonella Typhi and Salmonella Paratyphi A DNA from potential sources of transmission. We collected 370, 1L drinking water samples from a population-based random sample of households in the Kathmandu and Kavre Districts of Nepal between February and October 2019. Between November 2019 and July 2021, we collected 380, 50mL river water samples from 19 sentinel sites on a monthly interval along the rivers leading through the Kathmandu and Kavre Districts. We processed drinking water samples using a single qPCR and processed river water samples using differential centrifugation and qPCR at 0 and after 16 hours of liquid culture enrichment. A 3-cycle threshold (Ct) decrease of Salmonella Typhi or Salmonella Paratyphi, pre- and post-enrichment, was used as evidence of growth. We also performed structured observations of human-environment interactions to understand pathways of potential exposure. RESULTS: Among 370 drinking water samples, Salmonella Typhi was detected in 7 samples (1.8%) and Salmonella Paratyphi A was detected in 4 (1.0%) samples. Among 380 river water samples, Salmonella Typhi was detected in 171 (45%) and Salmonella Paratyphi A was detected in 152 (42%) samples. Samples located upstream of the Kathmandu city center were positive for Salmonella Typhi 12% of the time while samples from locations in and downstream were positive 58% and 67% of the time respectively. Individuals were observed bathing, washing clothes, and washing vegetables in the rivers. IMPLICATIONS: These results suggest that drinking water was not the dominant pathway of transmission of Salmonella Typhi and Salmonella Paratyphi A in the Kathmandu Valley in 2019. The high degree of river water contamination and its use for washing vegetables raises the possibility that river systems represent an important source of typhoid exposure in Kathmandu.

Antibiotics Use among Geriatric Patients Admitted in the Department of Medicine in a Tertiary Care Centre: A Descriptive Cross-sectional Study.

Introduction: Ageing predisposes to increased risk of infections which make these population vulnerable to high risk of various chronic co-morbidities, organ dysfunction and mortality. Increased frequency of infections has led to an increasing proportion of geriatric patient admission to hospitals, and antibiotics therapy has long been recognized as a cornerstone in the treatment of infections. The aim of this study was to find out the prevalence of antibiotic use among geriatric patients admitted to the Department of Medicine in a tertiary care centre. Methods: A descriptive cross-sectional study was conducted among geriatric patients admitted to Department of Medicine in a tertiary care centre from 1 May 2022 to 31 August 2022. Ethical approval was obtained from the Institutional Review Committee (Reference number: 17/22). Patients with the age of ≥60 years, admitted to the Department of Medicine who stayed for at least 24 hours was included as the study population. Convenience sampling method was used. Point estimate and 95% Confidence Interval were calculated. Results: Among 520 geriatric patients, antibiotics was used in 252 (48.46%) (44.16-52.76, 95% Confidence Interval) patients. Ceftriaxone was the most common antibiotic used in 165 (65.48%) patients, followed by oral azithromycin in 72 (28.57%). The mean antibiotics used per patient was 1.59±0.73. Conclusions: The prevalence of antibiotic use in the geriatric population was found to be lower than in the other studies done in similar settings. Keywords: aged; antibiotics; drug utilization.

Investigating the Efficacy and Safety of Diacerein in the Management of Knee Osteoarthritis with reference to its conventional management.

BACKGROUND: Designated as a "priority disease" by World Health Organization, Osteoarthritis is the most common chronic rheumatic disease. Providing a proper treatment for Osteoarthritis is still a major public health challenge. Diacerein has been proposed as a slow acting, symptom modifying or even disease modifying drug used in Osteoarthritis having a risk-benefit ratio far better than conventionally used drugs. However, the evidence of efficacy and safety of use of Diacerein in Osteoarthritis is yet to be explored. Hence, this study attempted to investigate the efficacy and safety of Diacerein in the management of knee osteoarthritis. METHODS: This is an analytical cohort study comparing Diacerein with Non-steroidal anti-inflammatory drugs for two months in the management of knee OA. Efficacy was assessed by scores of Lysholm Knee Scoring Scale, Knee injury and Osteoarthritis Outcome Score - Physical Function Short form and Western Ontario and McMaster Universities Osteoarthritis Index. RESULTS: After two months of treatment, the post- treatment scores were significantly superior to the baseline scores in both the treatment groups (p<0.001). There were no significant differences among the post-treatment scores in two different treatment groups (p>0.05). Discoloration of urine and gastritis were the frequently reported adverse effects in Diacerein treatment group and Non-steroidal anti-inflammatory drugs treatment group respectively. CONCLUSIONS: Our findings have shown Diacerein is as effective as Non-steroidal anti-inflammatory drugs in treating knee OA patients. Diacerein was generally well tolerated, with a good safety profile. These findings indicate the need for further studies with experimental study design in larger scale.

Use of Unannounced Standardized Patient to Assess Quality of Care in Community Pharmacies / Medicine Shops: A Pilot, Cross-Sectional Study.

BACKGROUND: Community pharmacies are the most accessible healthcare providers l which plays a key role in primary healthcare services managing minor ailment and ensure the quality use of drugs. Our study aimed to assess the quality care from community pharmacies using unannounced standardized patient. METHODS: All community pharmacies from three municipalities of Kavrepalanchowk district were visited by unannounced standardized patients presenting with acute dysentery, seasonal influenza, acute gastritis, acute diarrhea and pulmonary tuberculosis. Responses were audio-recorded and checked using standard checklist. Descriptive analysis was performed and data were presented as frequencies and percentages. RESULTS: A total of 40 visits were performed for each case of acute dysentery, seasonal influenza and acute gastritis and 41 visits for acute diarrhea and pulmonary tuberculosis. During visits, on average, 17.7% (±12.3) of recommended questions were asked by the community pharmacies. Among the drug dispensed, on average, 1.9 (± 0.6) drugs were dispensed. All the community pharmacies 40 (100.0%) provided correct drugs in acute gastritis followed by 34 (85.0%) in acute dysentery, 31 (77.5%) in acute diarrhea and 21 (52.5) in seasonal influenza, whereas no pharmacies provided correct drug in the case of pulmonary TB. None of the pharmacies counseled on potential adverse effects. CONCLUSIONS: The study showed a high rate of drug dispensed without sufficient inquiry of the recommended symptoms for proper diagnosis and counseling regarding drug use was low. The study recommends a need for continuous training by concerned bodies to improve the quality of professional practice in the community pharmacies.

Antimicrobial Resistance in E. coli Isolated from Chicken Cecum Samples and Factors Contributing to Antimicrobial Resistance in Nepal.

Microorganisms with antimicrobial resistance (AMR) are prevalent among humans and animals, and also found in the environment. Though organisms with AMR can spread to humans via food from animal sources, the burden of AMR in food-producing animals remains largely unknown. Thus, we assessed the resistance pattern among Escherichia coli isolated from chicken cecum samples and explored issues contributing to AMR in animals in the Dhulikhel Municipality of Nepal. We conducted a mixed-methods study, comprising a cross-sectional quantitative component, with collection of chicken cecal samples from slaughter houses/shops. In addition, a descriptive qualitative component was undertaken, with a focus group discussion and key informant interviews among stakeholders involved in animal husbandry. Of the 190 chicken cecum samples collected, 170 (89%) were subjected to culture and drug sensitivity testing, of which E. coli was isolated from 159 (94%) samples. Of the 159 isolates, 113 (71%) had resistance to ≥3 antimicrobial class. Resistance to tetracycline (86%) and ciprofloxacin (66%) were most prevalent. Overuse of antimicrobials, easy availability of antimicrobials, and lack of awareness among farmers about AMR were major issues contributing to AMR. The high prevalence of resistance among E. coli in chicken cecal samples calls for rational use of antimicrobials, educating farmers, and multi-sectoral coordination.

The Resistance Patterns in E. coli Isolates among Apparently Healthy Adults and Local Drivers of Antimicrobial Resistance: A Mixed-Methods Study in a Suburban Area of Nepal.

Evidence-based decision-making to combat antimicrobial resistance (AMR) mandates a well-built community-based surveillance system for assessing resistance patterns among commensals and pathogenic organisms. As there is no such surveillance system in Nepal, we attempted to describe the antimicrobial resistance pattern in E. coli isolated from the fecal samples of apparently healthy individuals in Dhulikhel municipality and also explored the local drivers of AMR. We used a mixed-method design with a cross-sectional quantitative component and a descriptive qualitative component, with focus group discussion and key informant interviews as the data collection method. Fecal samples were collected from 424 individuals randomly selected for the study. E. coli was isolated from 85.9% of human fecal samples, of which 14% were resistant to ≥3 class of antimicrobials (multidrug resistant). Of the 368 isolates, resistance to ampicillin (40.0%), tetracycline (20.7%) and cefotaxime (15.5%) were most prevalent. The major drivers of AMR were: lack of awareness of AMR, weak regulations on sales of antimicrobials, poor adherence to prescribed medications, and incomplete dosage due to financial constraints. These findings indicate the need for strict implementation of a national drug act to limit the over-the-counter sales of antimicrobials. Additionally, awareness campaigns with a multimedia mix are essential for educating people on AMR.

Estimating typhoid incidence from community-based serosurveys: a multicohort study.

BACKGROUND: The incidence of enteric fever, an invasive bacterial infection caused by typhoidal Salmonellae (Salmonella enterica serovars Typhi and Paratyphi), is largely unknown in regions without blood culture surveillance. The aim of this study was to evaluate whether new diagnostic serological markers for typhoidal Salmonella can reliably estimate population-level incidence. METHODS: We collected longitudinal blood samples from patients with blood culture-confirmed enteric fever enrolled from surveillance studies in Bangladesh, Nepal, Pakistan, and Ghana between 2016 and 2021 and conducted cross-sectional serosurveys in the catchment areas of each surveillance site. We used ELISAs to measure quantitative IgA and IgG antibody responses to hemolysin E and S Typhi lipopolysaccharide. We used Bayesian hierarchical models to fit two-phase power-function decay models to the longitudinal antibody responses among enteric fever cases and used the joint distributions of the peak antibody titres and decay rate to estimate population-level incidence rates from cross-sectional serosurveys. FINDINGS: The longitudinal antibody kinetics for all antigen-isotypes were similar across countries and did not vary by clinical severity. The seroincidence of typhoidal Salmonella infection among children younger than 5 years ranged between 58·5 per 100 person-years (95% CI 42·1-81·4) in Dhaka, Bangladesh, to 6·6 per 100 person-years (4·3-9·9) in Kavrepalanchok, Nepal, and followed the same rank order as clinical incidence estimates. INTERPRETATION: The approach described here has the potential to expand the geographical scope of typhoidal Salmonella surveillance and generate incidence estimates that are comparable across geographical regions and time. FUNDING: Bill & Melinda Gates Foundation. TRANSLATIONS: For the Nepali, Bengali and Urdu translations of the abstract see Supplementary Materials section.

Distinct Histone Post-translational Modifications during Plasmodium falciparum Gametocyte Development.

Histones are the building units of nucleosomes, which constitute chromatin. Histone post-translational modifications (PTMs) play an essential role in epigenetic gene regulation. The Plasmodium falciparum genome encodes canonical and variant histones and a collection of conserved enzymes for histone PTMs and chromatin remodeling. Herein, we profiled the P. falciparum histone PTMs during the development of gametocytes, the obligatory stage for parasite transmission. Mass spectrometric analysis of histones extracted from the early, middle, and late stages of gametocytes identified 457 unique histone peptides with 90 PTMs, of which 50% were novel. The gametocyte histone PTMs display distinct patterns from asexual stages, with many new methylation sites in histones H3 and H3.3 (e.g., K14, K18, and K37). Quantitative analyses revealed a high abundance of acetylation in H3 and H4, mono-methylation of H3/H3.3 K37, and ubiquitination of H3BK112, suggesting that these PTMs play critical roles in gametocytes. Gametocyte histones also showed extensive and unique combinations of PTMs. These data indicate that the parasite harbors distinct transcription regulation mechanisms during gametocyte development and lay the foundation for further characterization of epigenetic regulation in the life cycle of the malaria parasite.

Anxiety, Perceived Stress and Coping Strategies among the Nepalese Nurses Working around the World During a Corona Virus Disease -19 Outbreak.

BACKGROUND: Nurses are the frontline health professional of healthcare delivery system prone to have psychosocial problems. This study aimed to explore anxiety, stress and coping strategies among the Nepalese Nurses working around the World during a corona virus disease -19 Outbreak. METHODS: A web based cross sectional study was conducted for a period of three month among 240 nurses from Nepal and working abroad. They were invited to participate via various web based networks. Anxiety Self rating scale, perceived stress Scale and coping strategies were used for data collection. Chisquare, spearman rho and Manwhitnney was used for data analysis. RESULTS: More than half 58.8% were <30 years of age,mean age was 31±7.29 ,range was 20-56 years.Only17.5%were having Mild to Extreme Anxiety and, 62.5% Nepalese nurses were having stress. Regarding coping strategies mean score is higher in positive reframing followed by acceptance.There was significant association between stress and demographic variables marital status and country.Nepalese Nurses working in Nepal were having more anxiety and stress mean score than Nepalese nurses working abroad. CONCLUSIONS: Nepalese nurses working in Nepal were having more anxiety and stress mean score than Nepalese nurses working in abroad. Mean score of coping strategies was higher in avoidant coping (Maladaptive coping) in nurses working in Nepal whereas mean score is higher in Approach coping (Adaptive coping) in Nepalese nurses working abroad.

Self-medication in Primary Dysmenorrhea among Undergraduate Students in a Medical College: A Descriptive Cross-sectional Study.

Introduction: Primary dysmenorrhea is painful menstruation in women with normal pelvic anatomy, usually beginning during adolescence, primarily associated with a normal ovulatory cycle. There is an increased likelihood of self-medication among medical students. The aim of this study is to find out the prevalence of self-medication in primary dysmenorrhea among undergraduate students in a medical college. Methods: A descriptive cross-sectional study was conducted in a medical college among undergraduate female students with primary dysmenorrhea from 1 February 2022 to 31 May 2022. Ethical approval was obtained from the Institutional Review Committee of the same institute (Reference number: 254/2021). Convenience sampling was done. Data were collected through a self-administered questionnaire. Point estimate and 95% Confidence Interval were calculated. Results: Among 213 students with primary dysmenorrhea, self-medication was found to be in 78 (36.62%) (30.15-43.09, 95% Confidence Interval)). Among all the self-medications used, mefenamic acid was most common, used by 45 (57.69%) students, followed by paracetamol 11 (14.10%). Conclusions: The prevalence of self-medication practice in primary dysmenorrhea among undergraduate students was lower when compared to similar studies done in similar settings. Keywords: dysmenorrhea; menstruation; prevalence; self medication.

Formulation and Characterization of Phytostanol Ester Solid Lipid Nanoparticles for the Management of Hypercholesterolemia: An ex vivo Study.

BACKGROUND: Phytostanols are naturally occurring compounds that reduce blood cholesterol levels significantly. However, their aqueous insolubility poses formulation challenges. AIM: To formulate and characterize solid lipid nanoparticle carriers for phytostanol esters to enhance the bioavailability of phytostanols. METHODS: Phytostanol ester solid lipid nanoparticles were formulated by the microemulsion method. They were characterized for particle size distribution, polydispersity index, shape, surface charge, entrapment efficiency, stability, chemical structure, and thermal properties. The uptake of the formulation by cell lines, HepG2 and HT-29, and its effect on cell viability were evaluated. RESULTS: The formulation of solid lipid nanoparticles was successfully optimised by varying the type of lipids and their concentration relative to that of surfactants in the present study. The optimised formulation had an average diameter of (171 ± 9) nm, a negative surface charge of (-23.0 ± 0.8) mV and was generally spherical in shape. We report high levels of drug entrapment at (89 ± 5)% in amorphous form, drug loading of (9.1 ± 0.5)%, nanoparticle yield of (67 ± 4)% and drug excipient compatibility. The biological safety and uptake of the formulations were demonstrated on hepatic and intestinal cell lines. CONCLUSION: Phytostanol ester solid lipid nanoparticles were successfully formulated and characterized. The formulation has the potential to provide an innovative drug delivery system for phytostanols which reduce cholesterol and have a potentially ideal safety profile. This can contribute to better management of one of the main risk factors of cardiovascular diseases.

Circulating Genotypes of Rotavirus Prior to Rotarix?vaccine Introduction in Kathmandu, Nepal.

BACKGROUND: In Nepal, it is estimated that about 3 million children under 5 years of age are prone to diarrhea and previous studies have shown rotavirus as the major etiological agent. Given the high burden of rotavirus, Rotarix® vaccine was introduced in the national immunization schedule in July 2020. This study was carried out in a tertiary health center from January- September 2018 to determine the burden of rotavirus diarrhea as well as genotypic variations in the circulating virus prior to vaccine introduction in Kathmandu, Nepal. METHODS: Hospital based cross sectional study was conducted among children less than 5 years of age attending Kanti Children's Hospital. Rotavirus antigen detection was performed by enzyme immunoassay using ProSpecT Rotavirus Microplate Assay. Rotavirus A positive samples were further confirmed by genotyping using Reverse-Transcription Polymerase Chain Reaction. RESULTS: A total of 530 children that included 184 males and 346 females were enrolled in this study. Rotavirus antigen was detected in 112 (21.1%) stool samples. Of the total 112 positive EIA stool samples that were genotyped, G12P[6] (30.3%) was found to be the most common type, followed by G3P[8] (26.8%), mixed type (14.3%), and G1P[6] (13.4%). CONCLUSIONS: Continued surveillance should be carried out nationwide in Nepal to understand the effectiveness of the vaccination program and to report any new trends in the circulating genotypes.

Epigenetic reader complexes of the human malaria parasite, Plasmodium falciparum.

Epigenetic regulatory mechanisms are central to the development and survival of all eukaryotic organisms. These mechanisms critically depend on the marking of chromatin domains with distinctive histone tail modifications (PTMs) and their recognition by effector protein complexes. Here we used quantitative proteomic approaches to unveil interactions between PTMs and associated reader protein complexes of Plasmodium falciparum, a unicellular parasite causing malaria. Histone peptide pull-downs with the most prominent and/or parasite-specific PTMs revealed the binding preference for 14 putative and novel reader proteins. Amongst others, they highlighted the acetylation-level-dependent recruitment of the BDP1/BDP2 complex and identified an PhD-finger protein (PHD 1, PF3D7_1008100) that could mediate a cross-talk between H3K4me2/3 and H3K9ac marks. Tagging and interaction proteomics of 12 identified proteins unveiled the composition of 5 major epigenetic complexes, including the elusive TBP-associated-factor complex as well as two distinct GCN5/ADA2 complexes. Furthermore, it has highlighted a remarkable degree of interaction between these five (sub)complexes. Collectively, this study provides an extensive inventory of PTM-reader interactions and composition of epigenetic complexes. It will not only fuel further explorations of gene regulation amongst ancient eukaryotes, but also provides a stepping stone for exploration of PTM-reader interactions for antimalarial drug development.

Acute gastroenteritis associated with Rotavirus A among children less than 5 years of age in Nepal.

BACKGROUND: Rotavirus gastroenteritis is a major public health problem in Nepal. This study was conducted to obtain information associated with Rotavirus gastroenteritis and to perform genotyping of Rotavirus A. METHODS: Hospital based cross sectional study was conducted from January to December 2017 among children less than 5 years of age attending Kanti Children's Hospital and Tribhuvan University Teaching Hospital. Rotavirus A antigen detection was performed by Enzyme Linked Immunosorbent Assay (ELISA) using ProSpecT Rotavirus Microplate Assay. Rotavirus A positive strains were further confirmed by genotyping using Reverse-Transcription Polymerase Chain Reaction (RT-PCR). RESULTS: A total of 1074 stool samples were collected, of them 770 were hospitalized, and 304 were non-hospitalized cases. Rotavirus A infection was found in 28% of children with infection rate higher in hospitalized (34%) than in non-hospitalized (14%) children. Rotavirus A detection was higher in male (31%) than in female (24%), but this was statistically not significant (p > 0.05). Rotavirus A positivity was higher in children of age group 0-23 months, this result was statistically not significant (p > 0.05) with higher frequency found in the months of November, December, January, February and March (p < 0.05). On the basis of molecular analysis of Rotavirus A genotyping, G12P[6] (46.39%) was found to be the predominant followed by G1P[8] (35.05%), G3P[8] (7.21%) and G1P[6] (5.15%) while 4.12% was mixed infection and 1.03% was partially typed (p < 0.05). CONCLUSION: Rotavirus A infection occurred throughout the year, but the infection was significantly higher during the month of March. The higher frequency of rotavirus infection was observed among children of age group 0-23 months; however this was not found to be statistically significant. In this study, G12P[6] is predominant genotype observed. The results of genotyping are essential for the introduction of Rotavirus vaccine in Nepal.

Burden of bacterial upper respiratory tract pathogens in school children of Nepal.

INTRODUCTION: Respiratory tract infections are one of the most common human infections in all age group and important cause of mortality and morbidity worldwide. Most bacterial upper respiratory tract infections are vaccine preventable. This study aimed to determine the prevalence of carrier state of bacterial upper respiratory tract pathogens among school children. It also aimed to study their antibiograms. METHODS: The specimen from posterior pharyngeal wall and tonsils were collected from 204 participants on calcium alginate coated swabs (HiMedia). Isolates were identified by standard microbiological methods and tested for in vitro antibiotic susceptibility testing by modified Kirby-Bauer disc diffusion method. RESULTS: In this study, Streptococcus pneumoniae (16.6%) was the most common bacterial pathogen recovered, followed by Staphylococcus aureus (14.7%), ß-haemolytic streptococci (non-Group A) (8.8%), Streptococcus pyogenes (5.3%) and Corynebacterium diphtheriae (3.4%). The Gram negative bacteria were Klebsiella pneumoniae (4.9%), Haemophilus influenzae (3.4%) and Neisseria meningitidis (1.4%). Important findings in antibiogram include high resistance of Streptococcus pneumoniae to penicillin (91.17%) and resistance of S. aureus to oxacillin (23.3%). CONCLUSION: Pharyngeal colonisation by S. pneumoniae was found high among school children and this calls for an urgent need to include pneumococcal vaccine in routine national immunisation schedule of Nepal given the high burden of invasive pneumococcal disease. Despite expected universal vaccination, pharyngeal colonisation by C. diphtheriae is possible and there is possibility of transmission of these respiratory pathogens to other healthy children.

Sex-Specific Biology of the Human Malaria Parasite Revealed from the Proteomes of Mature Male and Female Gametocytes.

The gametocytes of the malaria parasites are obligate for perpetuating the parasite's life cycle through mosquitoes, but the sex-specific biology of gametocytes is poorly understood. We generated a transgenic line in the human malaria parasite Plasmodium falciparum, which allowed us to accurately separate male and female gametocytes by flow cytometry. In-depth analysis of the proteomes by liquid chromatography-tandem mass spectrometry identified 1244 and 1387 proteins in mature male and female gametocytes, respectively. GFP-tagging of nine selected proteins confirmed their sex-partitions to be agreeable with the results from the proteomic analysis. The sex-specific proteomes showed significant differences that are consistent with the divergent functions of the two sexes. Although the male-specific proteome (119 proteins) is enriched in proteins associated with the flagella and genome replication, the female-specific proteome (262 proteins) is more abundant in proteins involved in metabolism, translation and organellar functions. Compared with the Plasmodium berghei sex-specific proteomes, this study revealed both extensive conservation and considerable divergence between these two species, which reflect the disparities between the two species in proteins involved in cytoskeleton, lipid metabolism and protein degradation. Comparison with three sex-specific proteomes allowed us to obtain high-confidence lists of 73 and 89 core male- and female-specific/biased proteins conserved in Plasmodium The identification of sex-specific/biased proteomes in Plasmodium lays a solid foundation for understanding the molecular mechanisms underlying the unique sex-specific biology in this early-branching eukaryote.

Genome-wide association analysis identifies genetic loci associated with resistance to multiple antimalarials in Plasmodium falciparum from China-Myanmar border.

Drug resistance has emerged as one of the greatest challenges facing malaria control. The recent emergence of resistance to artemisinin (ART) and its partner drugs in ART-based combination therapies (ACT) is threatening the efficacy of this front-line regimen for treating Plasmodium falciparum parasites. Thus, an understanding of the molecular mechanisms that underlie the resistance to ART and the partner drugs has become a high priority for resistance containment and malaria management. Using genome-wide association studies, we investigated the associations of genome-wide single nucleotide polymorphisms with in vitro sensitivities to 10 commonly used antimalarial drugs in 94 P. falciparum isolates from the China-Myanmar border area, a region with the longest history of ART usage. We identified several loci associated with various drugs, including those containing pfcrt and pfdhfr. Of particular interest is a locus on chromosome 10 containing the autophagy-related protein 18 (ATG18) associated with decreased sensitivities to dihydroartemisinin, artemether and piperaquine - an ACT partner drug in this area. ATG18 is a phosphatidylinositol-3-phosphate binding protein essential for autophagy and recently identified as a potential ART target. Further investigations on the ATG18 and genes at the chromosome 10 locus may provide an important lead for a connection between ART resistance and autophagy.

A field-deployable mobile molecular diagnostic system for malaria at the point of need.

In response to the urgent need of a field-deployable and highly sensitive malaria diagnosis, we developed a standalone, "sample-in-answer-out" molecular diagnostic system (AnyMDx) to enable quantitative molecular analysis of blood-borne malaria in low resource areas. The system consists of a durable battery-powered analyzer and a disposable microfluidic compact disc loaded with reagents ready for use. A low power thermal module and a novel fluorescence-sensing module are integrated into the analyzer for real-time monitoring of loop-mediated isothermal nucleic acid amplification (LAMP) of target parasite DNA. With 10 µL of raw blood sample, the AnyMDx system automates the nucleic acid sample preparation and subsequent LAMP and real-time detection. Under laboratory conditions with whole-blood samples spiked with cultured Plasmodium falciparum, we achieved a detection limit of ∼0.6 parasite per µL, much lower than those for the conventional microscopy and rapid diagnostic tests (∼50-100 parasites per µL). The turnaround time from sample to answer is less than 40 minutes. The AnyMDx is user-friendly requiring minimal technological training. The analyzer and the disposable reagent compact discs are cost-effective, making AnyMDx a potential tool for malaria molecular diagnosis under field settings for malaria elimination.

Growing Menace of Antibacterial Resistance in Clinical Isolates of Pseudomonas aeruginosa in Nepal: An Insight of Beta-Lactamase Production.

Introduction. Pseudomonas aeruginosa is the most frequently isolated organism as it acts as the opportunistic pathogen and can cause infections in immunosuppressed patients. The production of different types of beta-lactamases renders this organism resistant to many commonly used antimicrobials. Therefore, the aim of this study was to document the antibiotic resistance rate in Pseudomonas aeruginosa isolated from different clinical specimens. Methods. Pseudomonas aeruginosa recovered was identified by standard microbiological methods. Antibiotic susceptibility testing was performed by modified Kirby-Bauer disc diffusion method following Clinical and Laboratory Standard Institute (CLSI) guidelines and all the suspected isolates were tested for the production of ESBLs, MBLs, and AmpC. Results. Out of total (178) isolates, 83.1% were recovered from the inpatient department (IPD). Majority of the isolates mediated resistance towards the beta-lactam antibiotics, while nearly half of the isolates were resistant to ciprofloxacin. Most of the aminoglycosides used showed resistance rate up to 75% but amikacin proved to be better option. No resistance to polymyxin was observed. ESBLs, MBLs, and AmpC mediated resistance was seen in 33.1%, 30.9%, and 15.7% isolates, respectively. Conclusions. Antibiotic resistance rate and beta-lactamase mediated resistance were high. Thus, regular surveillance of drug resistance is of utmost importance.