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Water distribution systems are one of the critical infrastructures and major assets of the water utility in a nation. The infrastructure of the distribution systems consists of resources, treatment plants, reservoirs, distribution lines, and consumers. A sustainable water distribution network management has to take care of accessibility, quality, quantity, and reliability of water. As water is becoming a depleting resource for the coming decades, the regulation and accounting of water in terms of the above four parameters is a critical task. There have been many efforts towards the establishment of a monitoring and controlling framework, capable of automating various stages of the water distribution processes. The current trending technologies such as Information and Communication Technology (ICT), Internet of Things (IoT), and Artificial Intelligence (AI) have the potential to track this spatially varying network to collect, process, and analyze the water distribution network attributes and events. In this work, we investigate the role and scope of the IoT technologies in different stages of the water distribution systems. Our survey covers the state-of-the-art monitoring and control systems for the water distribution networks, and the status of IoT architectures for water distribution networks. We explore the existing water distribution systems, providing the necessary background information on the current status. This work also presents an IoT Architecture for Intelligent Water Networks - IoTA4IWNet, for real-time monitoring and control of water distribution networks. We believe that, these components need to be designed and implemented effectively to build a robust water distribution network.

Various international organizations involved in meteorology sponsored the International Symposium on Indian Monsoon and Pollution over the Monsoon Environment held in New Delhi, India, on Dec 2-5, 1997. The symposium, organized by the Indian Meteorological Society, focused on monsoon studies, atmospheric pollution and their subsequent forecast models. The meeting also delved on the utilization and significance of satellite data and meteorological instruments.

Background: Sepsis claims 1 in 5 lives annually as per global statistics. Sepsis incidence in recent studies represents at least 35 % of all ICU admissions and has a high mortality rate, especially in the presence of co-existing morbidities. The challenge has been to accurately diagnose the causative organism, considering factors such as possible polymicrobial infections, commensals and environmental contaminants. Legacy techniques such as culture, automated culture systems or even newer species-specific PCR or film array these challenges difficult to overcome. The Bactfast® and Fungifast® assays along with the integrated workflow is based on next generation sequencing and have the ability to demarcate infecting pathogen from contamination and commensal. The unique ability to pinpoint the exact pathogen, considering the commensal and contamination in a variety of samples, with an extremely high sensitivity could lead it to be a tool of diagnostic choice for non-resolving ICU sepsis due to its comprehensive coverage and speed. The aim of this study was to evaluate the use of Bactfast® and Fungifast® as a last mile diagnostic tool in a ICU setting., Method: This study was carried out considering access to four intensive care units (ICU). Legacy testing, mostly done on culture, was conducted at the various integrated microbiology facilities of the hospitals where the ICUs were located, in Chennai, India. NABL accredited laboratory Micro Genomics (India) Pvt Ltd, was established as the central processing facility for next generation sequencing to run the Bactfast® and Fungifast® assay. Co-relation of results for 490 samples was done retrospectively by a multi-disciplinary team of consultants which comprised of microbiologists, and infectious disease physicians., Result: The diagnostic workflow established with the Bactfast® assay provided a sensitivity of 94.1 % and specificity of 86.6 %. Identification of pathogens in Bactfast® was better when compared to the data published in 2017, as reflected by positive co-relation with clinical confirmation. Although the Fungifast® specificity was high, at 99.4 %, only 12 samples were positive on fungal culture out of 490 samples. Therefore, it was concluded a further study for fungi based on multiple technologies with more true positive samples is required to evaluate the test., Conclusion: Bactfast® can identify pathogens in a sample without any bias. Its introduction as diagnostic modality in life threatening ICU sepsis could reduce mortality and morbidity. Although the initial results of Fungifast® are encouraging a further research is required for more information on test sensitivity., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Financial: This study was sponsored by Micro Genomics India Pvt Ltd for the validation of Bactfast and Fungifast products of Credence Genomics Pvt Ltd. Author VSG is the Managing director of Micro Genomics India Pvt Ltd and Credence Genomics Pvt Ltd. Author AR is employee of Micro Genomics India Pvt Ltd. AM is past employee of Micro Genomics India Pvt Ltd. Author LMA is employee of Credence Genomics Pvt Ltd. SS, VP, TJ are past employee of Credence Genomics Pvt Ltd.Author VC is affiliated with Sri Lanka Institute of Biotechnology (SLIBTEC), Pitipana, Homagama, Sri Lanka.and past employee of Credence Genomics Pvt. Ltd. as a consultant in molecular biology at the time of the study. Author JP is a collaborator in the University of Colombo as the Professor of Microbiology, JP is also affiliated with Credence Genomics Pvt. Ltd. as a specialist in Medical Microbiology at the time of the study., (© 2024 The Authors. Published by Elsevier Ltd.)

Rhodococcus hoagii is a gram positive actinomycete found in horses and cattle. Humans can be infected by ingestion or inhalation through contaminated food or soil. The organism usually infects immunosuppressed hosts with pneumonia being the common presentation. We present a case of an 89 years old, apparently immunocompetent host presenting with fever, encephalopathy and arthritis who grew Rhodococcus hoagii in blood and synovial fluid, The patient responded well to a combination of vancomycin, azithromycin and imipenem-cilastatin. Our case demonstrates that extra-pulmonary manifestations such as septic arthritis and bacteremia can be seen in immune competent hosts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Background: The availability of rapid diagnostic platforms for positive blood cultures has accelerated the speed at which the clinical microbiology laboratory can identify the causative organism and facilitate early appropriate antimicrobial therapy. There is a paucity of data regarding the clinical utility of the blood culture identification 2 (BCID2) panel test and its correlation with phenotypic drug susceptibility testing (DST) in flagged blood culture bottles from intensive care units (ICUs) in countries such as India, which have high rates of multidrug-resistant gram-negative bacteria (MDR-GNB)., Materials and Methods: We conducted a retrospective observational study in a tertiary care ICU on 200 patients above 18 years of age in whom a BCID2 test was ordered when blood cultures flagged positive., Results: We found 99% concordance between BCID2 and cultures in the identification of bacteria and yeasts and 96.5% concordance between phenotypic and genotypic DST. Furthermore, BCID2 was available about 1.5 days earlier than conventional ID and DST and played a key role in tailoring antimicrobials in 82.5% of the patients. Polymyxin-based therapy was discontinued earlier after an empiric dose in 138 patients (69%) based on BCID2 reports., Conclusion: In critically ill patients with monomicrobial bacteremia, BCID2 rapidly identifies bacteria and antimicrobial resistance (AMR) genes and is significantly faster than conventional culture and sensitivity testing. Antibiotics were escalated in more than a third of patients and de-escalated in almost a fifth on the same day. We recommend that all ICUs routinely incorporate the test in their antibiotic decision-making process and in antimicrobial stewardship., How to Cite This Article: Vineeth VK, Nambi PS, Gopalakrishnan R, Sethuraman N, Ramanathan Y, Chandran C, et al. Clinical Utility of Blood Culture Identification 2 Panel in Flagged Blood Culture Samples from the Intensive Care Unit of a Tertiary Care Hospital. Indian J Crit Care Med 2024;28(5):461-466., Competing Interests: Source of support: Nil Conflict of interest: None, (Copyright © 2024; The Author(s).)

Background: There is a scarcity of data regarding nosocomial infections in patients with COVID-19 treated with ECMO. This observational study from India aims to describe the epidemiology and microbiology of infections in patients with COVID-19 associated ECMO., Methods: This is an ambi-directional observational study of COVID-19 ECMO patients admitted from April 2021 to June 2022 in a tertiary care hospital. The total number of sepsis episodes for each patient was recorded and were categorized as bloodstream infections (BSI), pneumonias, skin and soft tissue infections (SSTI), invasive candidiasis (IC), catheter associated urinary tract infection (CAUTI), intra-abdominal infections (IAI), and Clostridioides difficile infections. Details regarding each infection including the microbiological profile and outcomes were recorded., Results: 29 patients who received ECMO for COVID-19 pneumonia during the study period were identified. Of the 29 patients, there were a total of 185 septic episodes. The incidence of septic episodes was 72.4 per 1000 ECMO days. Of the 185 sepsis events, 82 (44.3%) were BSI, 72 (39%) were pneumonia, 19 (10.3%) were SSTI, 7 (3.8%) were CAUTI and 5 (2.7%) were IAIs. Of these 29 patients, 16 (55.2%) patients were discharged and 13 (44.8%) died., Conclusions: The most common infections in our patients were bloodstream infections followed by pneumonia. High rates of gram negative infections, including those caused by carbapenem resistant bacteria, reflect the Indian critical care unit epidemiology in general. Despite these high infection rates with antimicrobial resistant set of micro-organisms, we had a successful outcome in 55.2% of patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Objectives: To compare COVID-19-associated pulmonary mucormycosis (CAPM) with COVID-19-associated rhino-orbital mucormycosis (CAROM), ascertain factors associated with CAPM among patients with COVID-19, and identify factors associated with 12-week mortality in CAPM., Methods: We performed a retrospective multicentre cohort study. All study participants had COVID-19. We enrolled CAPM, CAROM, and COVID-19 subjects without mucormycosis (controls; age-matched). We collected information on demography, predisposing factors, and details of COVID-19 illness. Univariable analysis was used to compare CAPM and CAROM. We used multivariable logistic regression to evaluate factors associated with CAPM (with hypoxemia during COVID-19 as the primary exposure) and at 12-week mortality., Results: We included 1724 cases (CAPM [n = 122], CAROM [n = 1602]) and 3911 controls. Male sex, renal transplantation, multimorbidity, neutrophil-lymphocyte ratio, intensive care admission, and cumulative glucocorticoid dose for COVID-19 were significantly higher in CAPM than in CAROM. On multivariable analysis, COVID-19-related hypoxemia (aOR, 2.384; 95% CI, 1.209-4.700), male sex, rural residence, diabetes mellitus, serum C-reactive protein, glucocorticoid, and zinc use during COVID-19 were independently associated with CAPM. CAPM reported a higher 12-week mortality than CAROM (56 of the 107 [52.3%] vs. 413 of the 1356 [30.5%]; p = 0.0001). Hypoxemia during COVID-19 (aOR [95% CI], 3.70 [1.34-10.25]) and Aspergillus co-infection (aOR [95% CI], 5.40 [1.23-23.64]) were independently associated with mortality in CAPM, whereas surgery was associated with better survival., Discussion: CAPM is a distinct entity with a higher mortality than CAROM. Hypoxemia during COVID-19 illness is associated with CAPM. COVID-19 hypoxemia and Aspergillus co-infection were associated with higher mortality in CAPM., (Copyright © 2023 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Introduction and Background: Rapid molecular diagnostics to predict carbapenem resistance well before the availability of routine drug sensitivity testing (DST) can serve as an antimicrobial stewardship tool in the context of high rates of Carbapenem-resistant Enterobacteriaceae (CRE)., Materials and Methods: A retrospective observational study of patients more than 18 years of age on whom Xpert Carba-R (FDA approved for rectal swab specimen) was done on gram-negative bacteria (GNB) flagged blood culture samples, in an Indian intensive care unit between January 2015 and November 2018. We analyzed the performance of Xpert Carba-R in comparison with routine DST., Results: A total of 164 GNBs were isolated from 160 patients. Klebsiella pneumoniae and Escherichia coli were the predominant isolates. Carba-R was positive in 35.36% of samples and 45.34% were carbapenem-resistant (CR) on routine DST. The distribution of the CR gene was: Oxacillinase (OXA) (50%), NDM (32.7%) followed by OXA and NDM co-expression (15.51%). The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value, and negative predictive value of Carba-R were 90.74, 93.15, 13.25, 0.10, 83.58 and 96.31% for Enterobacteriaceae . The median time to obtain the Carba-R report was 30 hours 34 minutes vs 74 hours and 20 minutes for routine DST. Based on the Carba-R report, 9.72% of patients had escalation and 27.08% had de-escalation of antibiotics., Conclusion: Xpert Carba-R serves as a rapid diagnostic tool for predicting carbapenem resistance in intensive care unit patients with bacteremia caused by Enterobacteriaceae ., How to Cite This Article: Rajendran S, Gopalakrishnan R, Tarigopula A, Kumar DS, Nambi PS, Sethuraman N, et al . Xpert Carba-R Assay on Flagged Blood Culture Samples: Clinical Utility in Intensive Care Unit Patients with Bacteremia Caused by Enterobacteriaceae. Indian J Crit Care Med 2023;27(9):655-662., Competing Interests: Source of support: Nil Conflict of interest: None, (Copyright © 2023; The Author(s).)

Biological therapeutic agents are highly targeted and potent but limited in their ability to reach intracellular targets. These limitations often necessitate high therapeutic doses and can be associated with less-than-optimal therapeutic activity. One promising solution for therapeutic agent delivery is use of cell-penetrating peptides. Canonical cell-penetrating peptides, however, are limited by low efficiencies of cellular uptake and endosomal escape, minimal proteolytic stability, and toxicity. To overcome these limitations, we designed a family of proprietary cyclic cell-penetrating peptides that form the core of our endosomal escape vehicle technology capable of delivering therapeutic agent-conjugated cargo intracellularly. We demonstrated the therapeutic potential of this endosomal escape vehicle platform in preclinical models of muscular dystrophy with distinct disease etiology. An endosomal escape vehicle-conjugated, splice-modulating oligonucleotide restored dystrophin protein expression in striated muscles in the mdx mouse, a model for Duchenne muscular dystrophy. Furthermore, another endosomal escape vehicle-conjugated, sterically blocking oligonucleotide led to knockdown of aberrant transcript expression levels in facioscapulohumeral muscular dystrophy patient-derived skeletal muscle cells. These findings suggest a significant therapeutic potential of our endosomal escape vehicle conjugated oligonucleotides for targeted upregulation and downregulation of gene expression in neuromuscular diseases, with possible broader application of this platform for delivery of intracellular biological agents., Competing Interests: X.L., M.K., P.G.D., K.J.K., X.S., N.L.E., S.P., A.P., S.L.B., E.S., M.D., M.G., N.S., and Z.Q. are employees of and hold stock in Entrada Therapeutics., (© 2023 The Author(s).)

Streptococcus pyogenes (SP) causes uncomplicated infections of throat & skin to severe life-threatening invasive diseases and poststreptococcal sequelae. Despite being common, it hasn't been studied much in recent times. Data of 93 adult patients >18 years, culture proven (SP) infections from 2016 to 2019 was studied in south India. Irrespective of comorbidities, SSTI were most common followed by surgical site infections& bacteremia. Isolates were susceptible to penicillin, cephalosporins but 23% were resistant to clindamycin. Timely surgical interventions and appropriate antibiotics reduced morbidity& limb salvage by 9 times. Larger studies, worldwide, to see the current trend of SP need to be conducted., (Copyright © 2022 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Bacground: Microbial Identification was done by phenotypic methods. VITEK-2 and Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) are now being increasingly used in laboratories., Objectives: To compare and evaluate the usefulness of MALDI-TOF MS and VITEK-2 in routine microbial identification., Methods: The performances of MALDI-TOF MS and VITEK 2 were compared for identifying microorganisms., Results: MALDI- TOF MS and VITEK-2 correctly identified 96 % (96/100) and 97% (97/100) of the isolates upto the genus level., Conclusion: MALDI TOF MS and VITEK -2 gave comparable identification and error rates. The rapid reduction in turnaround time with MALDI TOF is a significant game-changer in the field of clinical microbiology., Funding: State Board of Medical Research (SBMR)., Competing Interests: Conflict of interest: None declared, (Copyright © The Author(s).)

During September-December 2020, we conducted a multicenter retrospective study across India to evaluate epidemiology and outcomes among cases of coronavirus disease (COVID-19)-associated mucormycosis (CAM). Among 287 mucormycosis patients, 187 (65.2%) had CAM; CAM prevalence was 0.27% among hospitalized COVID-19 patients. We noted a 2.1-fold rise in mucormycosis during the study period compared with September-December 2019. Uncontrolled diabetes mellitus was the most common underlying disease among CAM and non-CAM patients. COVID-19 was the only underlying disease in 32.6% of CAM patients. COVID-19-related hypoxemia and improper glucocorticoid use independently were associated with CAM. The mucormycosis case-fatality rate at 12 weeks was 45.7% but was similar for CAM and non-CAM patients. Age, rhino-orbital-cerebral involvement, and intensive care unit admission were associated with increased mortality rates; sequential antifungal drug treatment improved mucormycosis survival. The COVID-19 pandemic has led to increases in mucormycosis in India, partly from inappropriate glucocorticoid use.

Introduction: Invasive candidiasis (IC) is a major cause of morbidity and mortality in critically ill patients in the intensive care unit (ICU). In this study, we aim to analyze the clinical profile, species distribution, and susceptibility pattern of patients with IC. Methods: Case records of non-neutropenic patients ≥18 years of age with IC between January 2016 and June 2019 at a tertiary care referral hospital were analyzed. IC was defined as either candidemia or isolation of Candida species from a sterile site (such as CSF; ascitic, pleural, or pericardial fluid; or pus or tissue from an intraoperative sample) in a patient with clinical signs and symptoms of infection. Results: A total of 114 patients were analyzed, out of which 105 (92.1%) patients had bloodstream infection (BSI) due to Candida and 9 (7.9%) had IC identified from a sterile site. Central line-associated blood stream infection (27 patients, 23.6%) and a gastrointestinal source (30 patients, 26.3%) were the most common presumed sources for candidemia. The commonest species was Candida tropicalis 42 (36.8%), followed by Candida glabrata 20 (17.5%). Serum beta-D-glucan (BDG) was done only in 32 patients of the 114 (35.3%); among those who were tested, 5 (15.6%) had a BDG value of less than 80 pg/mL despite having Candida BSI. Fluconazole sensitivity was 69.5% overall. At 14 days after diagnosis of IC, 49.1% had recovered, with the remainder having an unfavorable outcome (32.4% had died and 18.4% had left against medical advice). Clinical significance: IC is a major concern in Indian ICUs, with a satisfactory outcome in only half of our patients. Serum BDG is a valuable test to diagnose blood culture-negative IC, but more studies are needed to determine its role in the exclusion of IC, as we had a small minority of patients with negative tests despite proven IC. Conclusion: We recommend sending two sets of blood cultures and serum BDG assay for all suspected patients. Initiating empiric antifungal therapy with an echinocandin is advisable, in view of increasing azole resistance and the emergence of Candida auris , with de-escalation to fluconazole for sensitive isolates after clinical stability and blood culture clearance. How to cite this article: Sridharan S, Gopalakrishnan R, Nambi PS, Kumar S, Sethuraman N, Ramasubramanian V. Indian J Crit Care Med 2021;25(3):267-272., Competing Interests: Source of support: Nil Conflict of interest: None, (Copyright © 2021; Jaypee Brothers Medical Publishers (P) Ltd.)

Objective: The use of matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI) directly on urine can significantly improve turnaround time for diagnosing urinary tract infection (UTI). The present study was planned to compare the performance of MALDI directly on urine samples with conventional urine culture and evaluate an algorithm using a combination of significant pyuria and MALDI directly on urine samples as a screening method for diagnosing UTI., Materials and Methods: A total of 1000 urine samples from patients with symptoms suggestive of UTIs were analysed. Urine microscopy, urine culture and MALDI were performed simultaneously on all the samples., Results: MALDI correctly identified the microorganisms in 73.83% monomicrobial samples. In culture showing a mixed growth of two and more than three organisms, MALDI was able to identify one microorganism in 27.58% and 15.78% of samples, respectively. There were no peaks by MALDI in 93.53% of 464 sterile samples. The sequential algorithm using urine microscopy and MALDI could correctly identify UTI in 66.23% cases., Conclusion: MALDI can be utilised to rule out bacteriuria in >93% of sterile urine samples. The combination of significant pyuria and direct MALDI as screening method to diagnose UTI (whether monomicrobial or polymicrobial) was not found to be superior than using direct MALDI on urine samples alone., Competing Interests: None

Background: The (1,3)-β-D-glucan assay (BDG) is recommended for the early diagnosis of invasive candidiasis (IC)., Methods: Records of 154 critically ill adults with suspected IC, on whom BDG was done, were analyzed. Patients were divided into three groups: Group A (confirmed IC), Group B (alternative diagnosis or cause of severe sepsis), and Group C (high candidal score and positive BDG [>80 pg/mL] but without a confirmed diagnosis of IC)., Results: Mean BDG levels were significantly higher in Group A ( n = 32) as compared to Group B ( n = 60) and Group C ( n = 62) (448.75 ± 88.30 vs. 144.46 ± 82.49 vs. 292.90 ± 137.0 pg/mL; P < 0.001). Discontinuation of empiric antifungal therapy based on a value <80 resulted in cost savings of 14,000 INR per day per patient., Conclusion: A BDG value of <80 pg/ml facilitates early discontinuation of empirical antifungal therapy, with considerable cost savings., Competing Interests: There are no conflicts of interest.

The study was planned to measure the reduction of the load of bacterial flora on the blood donor's arm quantitatively using a three step protocol of donor arm cleansing incorporating either 70% isopropyl alcohol (IPA) or 5% w/v povidone iodine (PVI, 0.5% w/v available iodine) or 4% chlorhexidine gluconate (CHG) with or without the addition of 5% dimethyl sufloxide (DMSO). Single blind randomized study after obtaining ethical clearance, using the Miles and Misra technique for quantification and matrix assisted laser desorption ionization-mass spectrometry for identification of colony morphotypes on blood donor's skin. The mean pre-cleansing colony forming units (CFUs) was 89,318 and mean post-cleansing CFUs was 132, with a mean reduction of 99.85% with a mean log reduction of 3.24 (95% CI 2.01-4.47) at a P value of <0.0001. The post-cleansing CFUs was reduced to zero in all 34 samples in the protocol using CHG with DMSO, in 23 of 31 samples in the protocol using PVI with DMSO and 19 of 29 samples in the protocol using IPA with DMSO. The difference in means of the reduction of CFUs in protocols using CHG with DMSO compared with protocols using PVI or IPA with DMSO and PVI or IPA without DMSO was statistically significant with P value of 0.006, 0.0009, 0.015 and 0.05 respectively. The enhanced cutaneous antisepsis effect of CHG when complimented with DMSO in presence of IPA using the three step protocol of donor arm cleansing could stimulate more research and utilization of this as an additional safety towards the prevention of the problem of bacterial contamination of blood and blood components., Competing Interests: Compliance with Ethical StandardsThe authors disclose no conflicts of interest and no financial funding from any commercial organisation. The study was funded by PGIMER, Chandigarh, India.

Background & Objectives: Aeromonas species have been reported to cause various illnesses in humans such as wound infections, septicaemia, peritonitis and pneumonia. Their role in causation of cholera-like illness is also being increasingly recognized. This retrospective study was done to know the presence of Aeromonas as a cause of acute diarrhoea in a tertiary care hospital and to find the common species of Aeromonas causing diarrhoea and their antibiotic susceptibility patterns., Methods: Fifty isolates of Aeromonas were obtained over a period of 15 yr from 2000 to 2014 from patients of suspected acute gastroenteritis resembling cholera. Biotyping was done for 35 of these isolates available in culture collection, based on a panel of 13 biochemical reactions. Antibiogram was put up for all of these isolates by disk diffusion methods and interpreted according to the Clinical and Laboratory Standards Institute guidelines., Results: Of the 50 patients of Aeromonas-related acute gastroenteritis, 13 (26%) had typical features of cholera with rice water stools and severe dehydration. Eight patients (16%) had dysentery-like picture. One patient died of severe dehydration and septicaemia. The most common species were found to be Aeromonas caviae (34%) followed by Aeromonas veronii biovar veronii (29%), Aeromonas veronii biovar sobria (26%) and Aeromonas hydrophila (9%). All tested isolates were uniformly susceptible to cefepime, amikacin, azithromycin and meropenem; 14 per cent were susceptible to amoxicillin, 32 per cent to nalidixic acid, 60 per cent to co-trimoxazole, 54 per cent to ciprofloxacin, 60 per cent to ofloxacin, 74 per cent to chloramphenicol, 76 per cent to ceftriaxone, 74 per cent to cefotaxime, 88 per cent to gentamicin and 86 per cent to furoxone., Interpretation & Conclusions: Aeromonas is an important, often neglected pathogen capable of causing a variety of gastrointestinal tract symptoms such as acute diarrhoea and dysentery and may even mimic cholera. It is, therefore, pertinent to recognize this pathogen as an important agent in the causation of severe diarrhoea.

Coagulase negative staphylococci (CoNS) have recently emerged as important pathogens causing nosocomial blood stream infections. To evaluate the prevalence of CoNS in cutaneous normal flora, skin swabs were collected from voluntary blood donors and processed for culture and identification using matrix assisted laser desorption ionisation-time of flight (MALDI-TOF). CoNS were isolated from 96% of blood donors, most commonly Staphylococcus hominis (86%), followed by Staphylococcus epidermidis (22%) and Staphylococcus haemolyticus (9%). There has been a shift in the prevalent species of CoNS in the community in India, from the earlier known S. epidermidis towards resistant species such as S. hominis and S. haemolyticus. Routine and rapid speciation of CoNS in clinical isolates with MALDI-TOF should be used effectively to manage these resistant species.

With a completely reengineered and humanized glycosylation pathway, glycoengineered Pichia pastoris has emerged as a promising production host for the manufacture of therapeutic glycoproteins. However, the extensive genetic modifications have also negatively affected the overall fitness levels of the glycoengineered host cells. To make glycoengineered Pichia strains more compatible with a scalable industrial fermentation process, we sought to identify genetic solutions to broadly improve cell robustness during fermentation. In this study, we report that mutations within the Pichia pastoris ATT1 (PpATT1) gene (a homolog of the Saccharomyces cerevisiae GAL4 [ScGAL4] transcriptional activator) dramatically increased the cellular fitness levels of glycoengineered Pichia strains. We demonstrate that deletion of the PpATT1 gene enabled glycoengineered Pichia strains to improve their thermal tolerance levels, reduce their cell lysis defects, and greatly improve fermentation robustness. The extension of the duration of fermentation enabled the PpATT1-modified glycoengineered Pichia strains to increase their product yields significantly without any sacrifice in product quality. Because the ATT1 gene could be deleted from any Pichia strains, including empty hosts and protein-expressing production strains alike, we suggest that the findings described in this study are broadly applicable to any Pichia strains used for the production of therapeutic proteins, including monoclonal antibodies, Fc fusions, peptides, hormones, and growth factors., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Pichia pastoris is a methylotropic yeast that has gained great importance as an organism for protein expression in recent years. Here, we report the expression of recombinant human erythropoietin (rhEPO) in glycoengineered P. pastoris. We show that glycosylation fidelity is maintained in fermentation volumes spanning six orders of magnitude and that the protein can be purified to high homogeneity. In order to increase the half-life of rhEPO, the purified protein was coupled to polyethylene glycol (PEG) and then compared to the currently marketed erythropoiesis stimulating agent, Aranesp(®) (darbepoetin). In in vitro cell proliferation assays the PEGylated protein was slightly, and the non-PEGylated protein was significantly more active than comparator. Pharmacodynamics as well as pharmacokinetic activity of PEGylated rhEPO in animals was comparable to that of Aranesp(®). Taken together, our results show that glycoengineered P. pastoris is a suitable production host for rhEPO, yielding an active biologic that is comparable to those produced in current mammalian host systems., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to α-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal α-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) (1)H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D (1)H and (13)C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide α-Man-(1 → 2)-β-Man-(1 → 2)-β-Man-(1 → 2)-α-Man-(1 → 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple β-linkages on the α-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide., (© The Author 2011. Published by Oxford University Press. All rights reserved.)

Helicobacter pylori is the major causative agent of Gastric carcinoma. Significance of the urease accessory interaction proteins are emphasized in colonization of human gastric mucosa and efficient infection of H. pylori. Here an attempt is made to explore the structure and properties of urease accessory interaction proteins from Helicobacter pylori J 99. The proteins chosen for the study are ureH, ureI, nikR, groL and flgS based on the interaction map available from STRING database. The above mentioned proteins do not have a comprehensive three dimensional structure. Hence the models were generated using PSI-BLAST (Position Specific Iterative-Blast) and MODELLER 9V8. Physicochemical characterization encompasses pI, EC, AI, II and GRAVY. Secondary structure was predicted using PSI-PRED. Functional characterization was done by SOSUI and DISULFIND Servers and refinement of structure was done using Ramachandran plot analysis. RMS-Z values were calculated using Q-MEAN Server and CHIMERA was used for molecular simulation studies. Plant defensins from Vigna radiata are successfully docked to the modeled structures and thus interaction could be possibly prevented. These results will pave way for further selective inhibition of H. pylori colonization and in vivo survival by employing plant defensins from Vigna radiata (VrD1 & VrD2). The work will prove that plant defensins provides anticancer relief too.

Speakers of English and Tamil differ widely in which relational roles they overtly express with a verb. This study provides new information about how speakers of these languages differ in their descriptions of the same scenes and how explicit mention of roles and other scene elements vary with the properties of the scenes themselves. Specifically, we find that English speakers, who in normal speech rely more on explicit mention of verb arguments, in fact appear to be more affected by the pragmatic manipulations used in this study than Tamil speakers. Additionally, although the mention of scene items increases with development in both languages, Tamil-speaking children mention fewer items than do English-speaking children, showing that the children know the structure of the language to which they are exposed.

The methylotrophic yeast Pichia pastoris has recently been engineered to express therapeutic glycoproteins with uniform human N-glycans at high titers. In contrast to the current art where producing therapeutic proteins in mammalian cell lines yields a final product with heterogeneous N-glycans, proteins expressed in glycoengineered P. pastoris can be designed to carry a specific, preselected glycoform. However, significant variability exists in fermentation performance between genotypically similar clones with respect to cell fitness, secreted protein titer, and glycan homogeneity. Here, we describe a novel, multidimensional screening process that combines high and medium throughput tools to identify cell lines producing monoclonal antibodies (mAbs). These cell lines must satisfy multiple selection criteria (high titer, uniform N-glycans and cell robustness) and be compatible with our large-scale production platform process. Using this selection process, we were able to isolate a mAb-expressing strain yielding a titer (after protein A purification) in excess of 1 g/l in 0.5-l bioreactors.

Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation. The reported cell lines produce complex glycoproteins with greater than 90% terminal sialylation. Finally, to demonstrate the utility of these yeast strains, functional recombinant erythropoietin was produced.

Protein-based drugs constitute about a quarter of new approvals with a majority being glycoproteins. Increasing use of glycoproteins, such as monoclonal antibodies, at high therapeutic doses is challenging current production capacity. Mammalian cell culture, which is currently the production system of choice for glycoproteins, has several disadvantages including high cost of goods, long cycle times and, importantly, limited control over glycosylation. In view of this, several expression systems are currently being explored as alternatives to mammalian cell culture, these include yeast, plant and insect expression systems. Each of these has different merits for the production of therapeutic glycoproteins and can lead to enhanced therapeutic efficiency.

As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class. Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells. Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation.

The development of efficient germ-line transformation technologies for mosquitoes has increased the ability of entomologists to find, isolate and analyze genes. The utility of the currently available systems will be determined by a number of factors including the behavior of the gene vectors during the initial integration event and their behavior after chromosomal integration. Post-integration behavior will determine whether the transposable elements being employed currently as primary gene vectors will be useful as gene-tagging and enhancer-trapping agents. The post-integration behavior of existing insect vectors has not been extensively examined. Mos1 is useful as a primary germ-line transformation vector in insects but is inefficiently remobilized in Drosophila melanogaster and Aedes aegypti. Hermes transforms D. melanogaster efficiently and can be remobilized in this species. This element is also useful for creating transgenic A. aegypti, but its mode of integration in mosquitoes results in the insertion of flanking plasmid DNA. Hermes can be remobilized in the soma of A. aegypti and transposes using a common cut-and-paste mechanism; however, the element does not remobilize in the germ line. piggyBac can be used to create transgenic mosquitoes and occasionally integrates using a mechanism other than a simple cut-and-paste mechanism. Preliminary data suggest that remobilization is infrequent. Minos also functions in mosquitoes and, like the other gene vectors, appears to remobilize inefficiently following integration. These results have implications for future gene vector development efforts and applications.

Background: Human immunodeficiency virus (HIV) infection and AIDS is threatening the survival of many nations. To evaluate ongoing interventional strategies and burden of illness estimates, valid data on the prevalence of HIV are required. Often, in the absence of community prevalence data, estimates are based on surrogate markers such as prevalence of HIV in antenatal clinics. Even though the antenatal prevalence of HIV is easier to measure and can be repeated for evaluation, it is important to establish the association between antenatal and community prevalence of sexually transmitted diseases (STDs) and HIV, so that the validity of the estimates can be verified., Methods: A 'probability proportional to size' cluster survey was conducted in three randomly selected districts of Tamil Nadu in India. The basic unit of the survey was households from rural and urban clusters. Adults 15-45 years of age from the selected households were eligible for recruitment. Demographic, behavioural and laboratory data were collected. Clinical examination was done to identify STD syndromes and blood, urine, vaginal/urethral and endocervical swabs were taken for laboratory diagnosis of STDs from the subjects. Direct smear examination for Trichomonas vaginalis; serological tests for syphilis, hepatitis B, HIV, herpes simplex virus 2, Chlamydia trachomatis; and culture of Neisseria gonorrhoeae and Haemophilus ducreyi were performed on the collected specimens. The data were analysed adjusting for cluster effect., Result: We selected and screened 1981 individuals (1157 women and 824 men) for STDs and HIV from 1114 households representing the 25 million projected adult population of Tamil Nadu. The overall community prevalence of STDs including HIV and hepatitis B in Tamil Nadu was 14.6% (CI: 14.1-15.1), and 8.3% (CI: 7.9-8.6) when HIV and hepatitis B were excluded. Community prevalence of HIV and hepatitis B infection was 1.8% (CI:1.7-1.9) and 5.3% (CI: 5.1-5.5), respectively. The distribution of HIV involved both rural and urban regions of Tamil Nadu. On clinical examination, at least one STD syndrome was noted in 486 (24.5%) of the women subjects; vaginal discharge was the most common and found in 421 women (38.4%)., Conclusion: The prevalence of STD and HIV in Tamil Nadu is higher than expected and has extended into the non-high risk population (generalized epidemic).