Serological and Molecular Detection of Toxoplasma gondii in Domestic Pigs Intended for Human Consumption and Potential Occupational Hazard to Pig Farmers in India.

Toxoplasma gondii, an important food-borne zoonotic parasite, poses a worldwide public health hazard. Domestic pigs are considered one of the main intermediate hosts in the zoonotic transmission of T. gondii. To date, seroepidemiological information on T. gondii in domestic pigs in India is very scarce, and there are no reports of occupational hazards to pig farmers in this country. Here, we aimed at estimating the occurrence of T. gondii (antibodies and parasite DNA) in slaughtered pigs and pig farmers in Central India. Seroprevalence was determined in 410 serum samples from slaughtered pigs and 103 sera from pig farmers using an in-house prepared antigen-based modified agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA), and indirect-fluorescent antibody test (IFAT). Anti-T. gondii IgG antibodies were detected in 200 pigs (up to 48.8%, confidence interval [95% CI]: 40.4-52.2) and 44 pig farmers (up to 42.7%, 95% CI: 35.6-47.3) using MAT, ELISA, and IFAT. Inter-rater agreement showed an excellent agreement (kappa κ = 0.9) among the different serological tests suggesting similar detection potential of these tests. Recently acquired infections in all seropositive subjects were determined using IgG avidity testing and polymerase chain reaction (PCR). IgG avidity showed that 20 (10.3%) of slaughtered pigs and 8 (19.5%) pig farmers had a recently acquired infection. PCR for B1 and 529 repeats was performed in the heart tissues of slaughtered pigs and the blood cells of pig farmers. T. gondii DNA was detected in 14 (7.2%) slaughtered pigs and 5 (12.2%) pig farmers. Univariate analysis revealed that adult animals (>1 year), cats and rodents on the farm, and outdoor access are common factors (p ≤ 0.05) associated with T. gondii infection in pigs. Our results indicate that T. gondii is widely distributed in slaughtered pigs and pig farmers at risk of infection, highlighting a potential zoonotic transmission and health risk to consumers.

The Evolution of Central Venous-to-arterial Carbon Dioxide Difference (PCO2 Gap) during Resuscitation Affects ICU Outcomes: A Prospective Observational Study.

Introduction: The usual methods of perfusion assessment in patients with shock, such as capillary refill time, skin mottling, and serial serum lactate measurements have many limitations. Veno-arterial difference in the partial pressure of carbon dioxide (PCO2 gap) is advocated being more reliable. We evaluated serial change in PCO2 gap during resuscitation in circulatory shock and its effect on ICU outcomes. Materials and methods: This prospective observational study included 110 adults with circulatory shock. Patients were resuscitated as per current standards of care. We recorded invasive arterial pressure, urine output, cardiac index (CI), PCO2 gap at ICU admission at 6, 12, and 24 hours, and various patient outcomes. Results: Significant decrease in PCO2 gap was observed at 6 h and was accompanied by improvement in serum lactate, mean arterial pressure, CI and urine output in (n = 61). We compared these patients with those in whom this decrease did not occur (n = 49). Mortality and ICU LOS was significantly lower in patients with low PCO2 gap, while more patients with high PCO2 gap required RRT. Conclusion: We found that a persistently high PCO2 gap at 6 and 12 h following resuscitation in patients with shock of various etiologies, was associated with increased mortality, need for RRT and increased ICU LOS. High PCO2 gap had a moderate discriminative ability to predict mortality. How to cite this article: Zirpe KG, Tiwari AM, Kulkarni AP, Vaidya HS, Gurav SK, Deshmukh AM, et al. The Evolution of Central Venous-to-arterial Carbon Dioxide Difference (PCO2 Gap) during Resuscitation Affects ICU Outcomes: A Prospective Observational Study. Indian J Crit Care Med 2024;28(4):349-354.

Serological and molecular detection of Toxoplasma gondii and Neospora caninum in free-ranging rats from Nagpur, India.

Toxoplasma gondii and Neospora caninum are cyst-forming coccidian parasites that infect both wild and domestic non-felids as intermediate hosts, with rodents serving as important reservoir hosts during their life cycles. This study was aimed at investigating T. gondii and N. caninum infections and identifying factors favouring T. gondii infection in free-ranging rats from India. A total of 181 rodents were trap-captured, and blood and brain samples were subsequently collected for serological and molecular examination of T. gondii and N. caninum. Antibodies against T. gondii and N. caninum were detected by MAT/NAT and IFAT in 13.8% (25/181) and 1.65% (3/181) of rodents, respectively. All three N. caninum samples positive by NAT/IFAT were also positive for ELISA, while for T. gondii, 19 of 25 MAT/IFAT positive samples were also positive for ELISA. The antibody titers (MAT/NAT/IFAT) of rodents seropositive for T. gondii ranged from 25 to 400, while those of rats seropositive for N. caninum ranged from 25 to 100. Also, using PCR, DNA from T. gondii (B1 gene) and N. caninum (NC5 gene) was found in 2.76% (5/181) of brain samples and 0.55% (1/181) of brain samples. All PCR positive samples were also seropositive. No mixed infections were observed in the serological and molecular detections. A Chi-square analysis revealed that older rats and rats living in urban areas are significantly associated with T. gondii infection; however, rodent species, gender, location, habitat types, and seasonality were statistically nonsignificant. Overall, this study demonstrated that T. gondii was widely distributed while N. caninum was less prevalent among free-ranging rats in the studied area.

Impact of an Antimicrobial Stewardship Program on Broad Spectrum Antibiotics Consumption in the Intensive Care Setting.

Background and objectives: Antibiotics are the most commonly exploited agents in intensive care units. An antimicrobial stewardship program (ASP) helps in the optimal utilization of antibiotics and prevents the development of antibiotic resistance. The aim of this study was to assess the impact of ASP on broad-spectrum antibiotic consumption in terms of defined daily dose (DDD) and days of therapy (DOT) before and after the implementation of an ASP. Materials and methods: It was a prospective, quasi-experimental, pre- and post-study. Group A consisted of 5 months of ASP data, ASP activities were implemented during the next 2 months and continued. Group B (post-ASP) data was collected for the next 5 months. Total and individual DDDs and DOTs of broad-spectrum antibiotics utilized were compared between group A and group B. Results: Total DDDs used per 100 patient bed days were reduced by 18.72% post-ASP implementation (103.46 to 84.09 grams). The total DOT per 100 patient bed days used was 90.91 vs 71.25 days (21.62% reduction). As per the WHO classification of antibiotics use, the watch category (43.4% vs 43.04%) as well as reserve category (56.6% vs 56.97%) used between the two groups were found similar. The average length of stay (8.9 ± 2 days) after ASP was found significantly lesser than baseline (10.8 ± 3.4 days) (p < 0.05), however, there was no significant change in mortality between the two groups. Conclusion: Antimicrobial stewardship program implementation may reduce overall antibiotic consumption both in terms of DDD and DOT. How to cite this article: Zirpe KG, Kapse US, Gurav SK, Tiwari AM, Deshmukh AM, Suryawanshi PB, et al. Impact of an Antimicrobial Stewardship Program on Broad Spectrum Antibiotics Consumption in the Intensive Care Setting. Indian J Crit Care Med 2023;27(10):737-742.

Incidence, Subtypes, Risk factors, and Outcome of Delirium: A Prospective Observational Study from Indian Intensive Care Unit.

Background: Delirium is a common, under-recognized, and often fatal condition in critically ill patients, characterized by acute disorder of attention and cognition. The global prevalence varies with a negative impact on outcomes. A paucity of Indian studies exists that have systematically assessed delirium. Objective: A prospective observational study designed to determine the incidence, subtypes, risk factors, complications, and outcome of delirium in Indian intensive care units (ICUs). Patients and methods: Among 1198 adult patients screened during the study period (December 2019-September 2021), 936 patients were included. The confusion assessment method score (CAM-ICU) and Richmond agitation sedation scale (RASS) were used, with additional confirmation of delirium by the psychiatrist/neurophysician. Risk factors and related complications were compared with a control group. Results: Delirium occurred in 22.11% of critically ill patients. The hypoactive subtype was the most common (44.9%). The risk factors recognized were higher age, increased acute physiology and chronic health evaluation (APACHE-II) score, hyperuricemia, raised creatinine, hypoalbuminemia, hyperbilirubinemia, alcoholism, and smoking. Precipitating factors included patients admitted on noncubicle beds, proximity to the nursing station, requiring ventilation, as well as the use of sedatives, steroids, anticonvulsants, and vasopressors. Complications observed in the delirium group were unintentional removal of catheters (35.7%), aspiration (19.8%), need for reintubation (10.6%), decubitus ulcer formation (18.4%), and high mortality (21.3% vs 5%). Conclusion: Delirium is common in Indian ICUs with a potential effect on length of stay and mortality. Identification of incidence, subtype, and risk factors is the first step toward prevention of this important cognitive dysfunction in the ICU. How to cite this article: Tiwari AM, Zirpe KG, Khan AZ, Gurav SK, Deshmukh AM, Suryawanshi PB, et al. Incidence, Subtypes, Risk factors, and Outcome of Delirium: A Prospective Observational Study from Indian Intensive Care Unit. Indian J Crit Care Med 2023;27(2):111-118.

Seroprevalence, risk factors, and serological cross-reactivity for diagnosis of Toxoplasma gondii and Neospora caninum infections in goats in India.

Toxoplasma gondii and Neospora caninum are genetically related cyst-forming protozoan parasites that cause reproductive failures in ruminants. Given the limited information on the epidemiology of these infections in goats in India, the study aimed to estimate the seroprevalence, assess antibody cross-reactivity for diagnosis, and identify associated risk factors. A total of 695 sera were evaluated for antibodies to T. gondii and N. caninum infections using Modified Agglutination Test (MAT for Toxoplasma)/Neospora agglutination test (NAT), Enzyme-linked immunosorbent assay (ELISA), and Indirect Fluorescent Antibody Test (IFAT for tachyzoite and bradyzoite stages). The seroprevalence rate of T. gondii and N. caninum infections was 56.9% and 10.9%, respectively. Inter-rater agreement (kappa value - κ) was calculated to assess agreements between various diagnostic assays, using the IFAT as the gold standard (for detecting both infections), the agreements for MAT/NAT (κ = 0.97) and the ELISA (κ = 0.95) were excellent. The acute infection among seropositive goats were determined using serological (IgG avidity test - measures the binding strength between IgG antibodies and parasite antigens) and molecular diagnoses (PCR for repetitive DNA sequences - Toxoplasma B1 gene: 131 bp and Neospora NC5 gene: 328 bp). Among seropositive goats ≥80% had high IgG avidity and <10% of animals had low IgG avidity antibodies for both infections. Most low IgG avidity goats were PCR positive for the TgB1 gene (94.4%) or Nc5 gene (85.7%). In the serological assays, we used different dilutions of test serum to rule out the cross-reactivity owing to similar antigenic makeup between these two parasites. When the serological cross-reactivity was analyzed using invasion assay at a serum titer of ≥200, more than 90% T. gondii positive sera showed host cell invasion of N. caninum and vice versa. Largely, the serological results indicate that cut-off serum dilution of ≥1:200 for ELISA and IFAT and ≥1:25 for MAT/NAT avoids serological cross-reactivity between T. gondii and N. caninum. Further, the Univariate and multivariate analyses showed that adult animals (>2 years), reservoir hosts, and extensive rearing systems are common risk factors for these infections. However, the history of abortion was identified as a significant risk factor for T. gondii infection. This study revealed that T. gondii and N. caninum infections are highly prevalent in this region and the use of an appropriate cut-off serum dilution is necessary to avoid cross-reactivity between these closely related parasites.

Noninvasive Respiratory Assist Devices in the Management of COVID-19-related Hypoxic Respiratory Failure: Pune ISCCM COVID-19 ARDS Study Consortium (PICASo).

Objective: To determine whether high-flow nasal oxygen (HFNO) or noninvasive ventilator (NIV) can avoid invasive mechanical ventilation (IMV) in COVID-19-related acute respiratory distress syndrome (ADRS), and the outcome predictors of these modalities. Design: Multicenter retrospective study conducted in 12 ICUs in Pune, India. Patients: Patients with COVID-19 pneumonia who had PaO2/FiO2 ratio <150 and were treated with HFNO and/or NIV. Intervention: HFNO and/or NIV. Measurements: The primary outcome was to assess the need of IMV. Secondary outcomes were death at Day 28 and mortality rates in different treatment groups. Main results: Among 1,201 patients who met the inclusion criteria, 35.9% (431/1,201) were treated successfully with HFNO and/or NIV and did not require IMV. About 59.5% (714/1,201) patients needed IMV for the failure of HFNO and/or NIV. About 48.3, 61.6, and 63.6% of patients who were treated with HFNO, NIV, or both, respectively, needed IMV. The need of IMV was significantly lower in the HFNO group (p <0.001). The 28-day mortality was 44.9, 59.9, and 59.6% in the patients treated with HFNO, NIV, or both, respectively (p <0.001). On multivariate regression analysis, presence of any comorbidity, SpO2 <90%, and presence of nonrespiratory organ dysfunction were independent and significant determinants of mortality (p <0.05). Conclusions: During COVID-19 pandemic surge, HFNO and/or NIV could successfully avoid IMV in 35.5% individuals with PO2/FiO2 ratio <150. Those who needed IMV due to failure of HFNO or NIV had high (87.5%) mortality. How to cite this article: Jog S, Zirpe K, Dixit S, Godavarthy P, Shahane M, Kadapatti K, et al. Noninvasive Respiratory Assist Devices in the Management of COVID-19-related Hypoxic Respiratory Failure: Pune ISCCM COVID-19 ARDS Study Consortium (PICASo). Indian J Crit Care Med 2022;26(7):791-797.

Toxoplasma gondii induces robust humoral immune response against cyst wall antigens in chronically infected animals and humans.

Toxoplasma gondii differentiation from proliferating tachyzoites into latent bradyzoites is central to pathogenesis and transmission. Strong humoral immune response has been reported against tachyzoite antigens, however, antibody-mediated response towards bradyzoite antigens is poorly characterized. This work aimed to study the humoral immune response towards bradyzoite and associated cyst wall antigens particularly CST1. The immunoreactivity of 404 goats, 88 sheep and 92 human sera to recombinant (CST1 and SRS9) and native proteins of encysted bradyzoite along with well-established tachyzoite antigens (SAG1 and GRA7) was determined using ELISA, Western blot and immunofluorescence analysis (IFA). ELISA results revealed nearly 50% of sera contain T. gondii specific antibodies. Results were further validated using Western blot and IFA. T. gondii positive sera predominantly recognized the cyst wall besides the known tachyzoite surface antigens. The presence of CST1 antibodies in seropositive samples were in line with the staining patterns which were consistent with CST localization. Notably, T. gondii IgM- IgG+ sera recognize the cyst wall whereas IgM + IgG-sera recognize tachyzoite antigens indicating acute infection consistent with presence of parasite DNA. The study demonstrates a strong humoral response against bradyzoite associated cyst wall antigens across naturally infected animals and humans. CST1 emerged as a key immunomodulatory antigen which may have direct implications for clinical immunodiagnostics.

A Real-world Study on Prescription Pattern of Fosfomycin in Critical Care Patients.

BACKGROUND: This study presents a real-world scenario for prescription pattern, efficacy, and safety data on the current clinical use of intravenous fosfomycin in critically ill patients in Indian settings. PATIENTS AND METHODS: This was a retrospective cohort study conducted for a period of 10 months among critically ill patients admitted to hospital's critical care unit. The primary objective of the study was to analyze the prescription pattern of intravenous fosfomycin, and the secondary objective was to evaluate the safety profile and patient outcomes. RESULTS: A total of 309 patients were enrolled, and they were diagnosed with bacteremia (45.3%), pneumonia (15.85%), septic shock (14.24%), and urinary tract infections (UTI) (13.91%). The average dose of fosfomycin given was 11.7 ± 4.06 gm/day. The average duration of the therapy was 4.85 ± 3.59 days with a median duration of 4 days. Fosfomycin was given at 8 hourly dosing frequency to maximum (45.6%) cases. Hypokalemia was the most observed adverse event. The overall survival was seen in 55% of patients. CONCLUSION: Our data suggest that UTI, infection caused by Escherichia coli, and a daily dose of >12 g were associated with better clinical outcomes. The overall survival of critically ill patients receiving fosfomycin was 55%. HOW TO CITE THIS ARTICLE: Zirpe KG, Mehta Y, Pandit R, Pande R, Deshmukh AM, Patil S, et al. A Real-world Study on Prescription Pattern of Fosfomycin in Critical Care Patients. Indian J Crit Care Med 2021;25(9):1055-1058.

Timing of Invasive Mechanical Ventilation and Mortality among Patients with Severe COVID-19-associated Acute Respiratory Distress Syndrome.

BACKGROUND: Severe acute respiratory distress syndrome associated with coronavirus disease-2019 (COVID-19) (CARDS) pneumonitis presents a clinical challenge as regards to the timing of intubation and ambiguity of outcome. There is a lack of clear consensus on when to switch patients from trials of noninvasive therapies to invasive mechanical ventilation. We investigated the effect of the timing of intubation from the time of admission on the clinical outcome of CARDS. AIM AND OBJECTIVE: The aim and objective was to analyze the effect of timing of intubation early (within 48 hours of admission to critical care unit) versus delayed (after 48 hours of admission to critical care unit) on mortality in severe CARDS patients. MATERIALS AND METHODS: A retrospective observational study performed in a 28-bedded COVID-19 intensive care unit of a tertiary care hospital in Pune, India. All patients admitted between April 1, 2020, and October 15, 2020, with confirmed COVID-19 (RT-PCR positive) requiring mechanical ventilation were included in the study. RESULTS: The primary outcome was in-hospital mortality. Among 2,230 patients that were admitted to the hospital, 525 required critical care (23.5%), invasive mechanical ventilation was needed in 162 patients and 147 (28%) of critical care admission were included in the study cohort after exclusion. Seventy-five patients (51%) were intubated within 48 hours of critical care admission (early group) and 72 (48.9%) were intubated after 48 hours of critical care admission (delayed group). With regards to the total of 147 included patients; male patients were 74.1% with a median age of 59 years (interquartile range, 51-68 years). Diabetes (44.9%) and hypertension (43.5%) were the most common comorbidities. Higher admission acute physiology and chronic health evaluation II scores and lower absolute lymphocyte count were observed in patients intubated within 48 hours. The early intubated group had a mortality of 60% whereas the same was observed as 77.7% in delayed intubation group, and this difference was statistically significant (p = 0.02). CONCLUSION: Current study concludes that early intubation is associated with improved survival rates in severe CARDS patients. HOW TO CITE THIS ARTICLE: Zirpe KG, Tiwari AM, Gurav SK, Deshmukh AM, Suryawanshi PB, Wankhede PP, et al. Timing of Invasive Mechanical Ventilation and Mortality among Patients with Severe COVID-19-associated Acute Respiratory Distress Syndrome. Indian J Crit Care Med 2021;25(5):493-498.

The Second- vs First-wave COVID-19: More of the Same or a Lot Worse? A Comparison of Mortality between the Two Waves in Patients Admitted to Intensive Care Units in Nine Hospitals in Western Maharashtra.

BACKGROUND: India, along with the rest of the world, faced the challenging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. The second wave in India lagged behind that in the Western world, due to different timing of seasons. There is scarce data about the differences between the two waves, for intensive care unit (ICU) patients. We present the data of 3,498 patients from 9 ICUs of western Maharashtra. MATERIALS AND METHODS: We collected prospective data of hospitalized, RT-PCR confirmed, coronavirus-2019 (COVID-19) patients, from nine tertiary centers, after institutional ethics committee (IEC) approval. Then, we segregated and analyzed the data of patients admitted to the ICU, for comorbidities, high-resolution computed tomography (HRCT) score, ventilatory support, etc. The primary outcomes were ICU and hospital mortality. We also performed multivariable analysis for predictors of ICU mortality. RESULTS: Overall, there were 3,498 ICU patients. In the first wave, 1,921 patients needed ICU admission, while in the second wave, 1,577 patients. Patients in the second wave had significantly higher ICU (26.1 vs 13.4%, p <0.001) and hospital mortality (29.9 vs 18.2%, p <0.001) and need for ventilatory support of any type. More patients received steroids during the second wave. On multivariable regression, male gender, ICU admission during the second wave, increasing HRCT score, and need for intubation and mechanical ventilation were significant predictors of ICU mortality. CONCLUSION: ICU patients admitted during the two waves were of the similar age, but there were more females, and more patients had comorbidities during the second wave. The ICU and hospital mortality were significantly higher during the second wave. HOW TO CITE THIS ARTICLE: Zirpe KG, Dixit S, Kulkarni AP, Pandit RA, Ranganathan P, Prasad S, et al. The Second- vs First-wave COVID-19: More of the Same or a Lot Worse? A Comparison of Mortality between the Two Waves in Patients Admitted to Intensive Care Units in Nine Hospitals in Western Maharashtra. Indian J Crit Care Med 2021; 25(12):1343-1348.

The Impact of Early Tracheostomy in Neurotrauma Patients: A Retrospective Study.

BACKGROUND: Although majority of neurotrauma patients require long term ventilatory support but the timing of tracheostomy in such patients is controversial. METHOD: This retrospective study was conducted at a Tertiary Care Hospital, Pune, India. Patients >18 years of age, who underwent percutaneous tracheostomy (PCT) from June 2010 to November 2014 at neurotrauma unit (NTU) of hospital, were included. Patients were divided in two groups according to the timing of tracheostomy, early tracheostomy (ET) group (≤5 days; N=100) and late tracheostomy (LT) group (>5 days; N=64). The nonparametric Mann-Whitney test, and Chi-square tests were used to compare these groups. RESULT: There were no significant differences between the groups in terms of age, sex, APACHE II and GCS Score. Patients in the ET group had a significantly shorter stay in the NTU compared to patients in the LT group (mean, 18 vs. 21.2 days, p=0.005), fewer mechanical ventilation days (mean, 8.1 vs. 11.7 days, P=0.000) and shorter length of stay in hospital (mean, 28.8 vs. 34.37 days, P=0.019). There was no difference between ET and LT groups in post PCT ventilator free days (mean, 8.2 vs. 9.4 days; P=0.094). Mortality rates in ET vs. LT groups were also comparable (35% vs. 29.7%; P=0.480). CONCLUSION: Results suggest that ET in neurotrauma patients might be associated with shorter length of stay in NTU and hospital, and shorter duration of mechanical ventilation however there was no mortality difference.

Regulation of Plasmodium falciparum†Origin Recognition Complex subunit 1 (PfORC1) function through phosphorylation mediated by CDK-like kinase PK5.

Plasmodium falciparum Origin Recognition Complex subunit 1 (PfORC1) has been implicated in DNA replication and var gene regulation. While the C-terminus is involved in DNA replication, the specific role of N-terminus has been suggested in var gene regulation in a Sir2-dependent manner. PfORC1 is localized at the nuclear periphery, where the clustering of chromosomal ends at the early stage of parasite development may be crucial for the regulation of subtelomeric var gene expression. Upon disassembly of telomeric clusters at later stages of parasite development, ORC1 is distributed in the nucleus and parasite cytoplasm where it may be required for its other cellular functions including DNA replication. The level of ORC1 decreases dramatically at the late schizont stage. The mechanisms that mediate regulation of PfORC1 function are largely unknown. Here we show, by the use of recombinant proteins and of transgenic parasites expressing wild type or mutant forms of ORC1, that phosphorylation of the PfORC1-N terminal domain by the cyclin-dependent kinase (CDK) PfPK5 abolishes DNA-binding activity and leads to changes in subcellular localization and proteasome-mediated degradation of the protein in schizonts. These results reveal that PfORC1 phosphorylation by a CDK is central to the regulation of important biological functions like DNA replication and var gene silencing.

Regulation of DNA replication proteins in parasitic protozoans: possible role of CDK-like kinases.

Regulatory roles of CDKs in fundamental processes including cell cycle progression and transcription are well conserved in metazoans. This family of proteins has undergone significant evolutionary divergence and specialization. Several CDK-like kinases have been identified and characterized in parasitic protozoans. However, clear functional role and physiological relevance of these proteins in protozoans still remain elusive. In continuation with the recent finding that CDK-like protein PfPK5 regulates important DNA replication protein like origin recognition complex subunit 1 in Plasmodium falciparum, here we have discussed the emerging significance of CDK1/2 homologs in DNA replication of parasitic protozoans. In fact, involvement of these proteins in crucial cellular processes projects them as potential drug targets. The possibilities that CDKs offer as potential therapeutic targets in controlling parasite progression have also been explored.

Functional dissection of proliferating-cell nuclear antigens (1 and 2) in human malarial parasite Plasmodium falciparum: possible involvement in DNA replication and DNA damage response.

Eukaryotic PCNAs (proliferating-cell nuclear antigens) play diverse roles in nucleic acid metabolism in addition to DNA replication. Plasmodium falciparum, which causes human malaria, harbours two PCNA homologues: PfPCNA1 and PfPCNA2. The functional role of two distinct PCNAs in the parasite still eludes us. In the present study, we show that, whereas both PfPCNAs share structural and biochemical properties, only PfPCNA1 functionally complements the ScPCNA mutant and forms distinct replication foci in the parasite, which PfPCNA2 fails to do. Although PfPCNA1 appears to be the primary replicative PCNA, both PfPCNA1 and PfPCNA2 participate in an active DDR (DNA-damage-response) pathway with significant accumulation in the parasite upon DNA damage induction. Interestingly, PfPCNA genes were found to be regulated not at the transcription level, but presumably at the protein stability level upon DNA damage. Such regulation of PCNA has not been shown in eukaryotes before. Moreover, overexpression of PfPCNA1 and PfPCNA2 in the parasite confers a survival edge on the parasite in a genotoxic environment. This is the first evidence of a PfPCNA-mediated DDR in the parasite and gives new insights and rationale for the presence of two PCNAs as a parasite survival strategy and its probable success.

Preparation and Evaluation of Diclofenac Sodium Tablet Coated with Polyelectrolyte Multilayer Film Using Hypromellose Acetate Succinate and Polymethacrylates for pH-Dependent, Modified Release Drug Delivery.

Polyelectrolyte multilayer (PEM) film formed due to the electrostatic interaction between oppositely charged polyelectrolytes is of considerable interest because of their potential applications as both drug carriers and surface-modifying agents. In this study, in vitro studies were carried out on polyelectrolyte complexes formulated with Eudragit E (EE) and hypromellose acetate succinate (HPMCAS). The complexes of EE and HPMCAS were formulated by non-stoichiometric method. The prepared IPCs were investigated using Fourier transform infrared spectroscopy. Diclofenac sodium (DS) tablets were prepared and were coated with polymer solution of HPMCAS and EE to achieve pH-dependent and sustained-release tablets. Tablets were evaluated for their physical characteristics and in vitro drug release. The results of pharmacokinetic studies in rabbits showed that the selected formulation (F6) exhibited a delayed peak plasma concentration and marked sustained-release effect of drug in the in vivo drug release in comparison with marketed tablet. The suitable combination of PEM film based on EE and HPMCAS demonstrated potential candidate for targeted release of DS in the lower part of the gastrointestinal (GI) tract.

Multilateral contracting and prevention.

Incentives created through contracts can be used as a means of decentralized control in healthcare systems to ensure more efficient healthcare. In this paper, we consider an insurer contracting with a consumer and a provider. We focus on the trade-off between ex ante moral hazard and insurance, and consider both consumer and provider incentives in the insurer's contracting problem in the presence of unobservable preventive efforts. We study two cases of provider efforts: those that complement consumer efforts and those that substitute for consumer efforts. In the first case, our results show that the provider must have greater incentives when the consumer is healthy to induce effort and that inducing provider effort allows an insurer to offer a more complete insurance contract relative to the bilateral benchmark. In the second case, we state conditions under which these conclusions continue to hold. On the basis of our findings, we discuss the implications and challenges of multilateral contracting in practice.

The role of N-terminus of Plasmodium falciparum ORC1 in telomeric localization and var gene silencing.

Plasmodium falciparum origin recognition complex 1 (ORC1) protein has been implicated in DNA replication and silencing var gene family. However, the mechanism and the domain structure of ORC1 related to the regulation of var gene family are unknown. Here we show that the unique N-terminus of PfORC1 (PfORC1N(1-238)) is targeted to the nuclear periphery in vivo and this region binds to the telomeric DNA in vitro due to the presence of a leucine heptad repeats. Like PfORC1N(1-238), endogenous full length ORC1, was found to be associated with sub telomeric repeat regions and promoters of various var genes. Additionally, binding and propagation of ORC1 to telomeric and subtelomeric regions was severely compromised in PfSir2 deficient parasites suggesting the dependence of endogenous ORC1 on Sir2 for var gene regulation. This feature is not previously described for Plasmodium ORC1 and contrary to yeast Saccharomyces cerevisiae where ORC function as a landing pad for Sir proteins. Interestingly, the overexpression of ORC1N(1-238) compromises the binding of Sir2 at the subtelomeric loci and var gene promoters consistent with de-repression of some var genes. These results establish role of the N-terminus of PfORC1 in heterochromatin formation and regulation of var gene expression in co-ordination with Sir2 in P. falciparum.

Plasmodium falciparum: epigenetic control of var gene regulation and disease.

Plasmodium falciparum, one of the deadliest parasites on earth causes human malaria resulting one million deaths annually. Central to the parasite pathogenicity and morbidity is the switching of parasite virulence (var) gene expression causing host immune evasion. The regulation of Plasmodium var gene expression is poorly understood. The complex life cycle of Plasmodium and mutually exclusive expression pattern of var genes make this disease difficult to control. Recent studies have demonstrated the pivotal role of epigenetic mechanism for control of coordinated expression of var genes, important for various clinical manifestations of malaria. In this review, we discuss about different Plasmodium histones and their various modifications important for gene expression and gene repression.Contribution of epigenetic mechanism to understand the var gene expression is also highlighted. We also describe in details P. falciparum nuclear architecture including heterochromatin, euchromatin and telomeric regions and their importance in subtelomeric and centrally located var gene expression. Finally, we explore the possibility of using Histone Acetyl Transferase (HAT) and Histone Deacetylase (HDAC)inhibitors against multi-drug resistance malaria parasites to provide another line of treatment for malaria.

CRISPR-Cas assisted diagnostics of plant viruses and challenges.

Plant viruses threaten global food security by infecting commercial crops, highlighting the critical need for efficient virus detection to enable timely preventive measures. Current techniques rely on polymerase chain reaction (PCR) for viral genome amplification and require laboratory conditions. This review explores the applications of CRISPR-Cas assisted diagnostic tools, specifically CRISPR-Cas12a and CRISPR-Cas13a/d systems for plant virus detection and analysis. The CRISPR-Cas12a system can detect viral DNA/RNA amplicons and can be coupled with PCR or isothermal amplification, allowing multiplexed detection in plants with mixed infections. Recent studies have eliminated the need for expensive RNA purification, streamlining the process by providing a visible readout through lateral flow strips. The CRISPR-Cas13a/d system can directly detect viral RNA with minimal preamplification, offering a proportional readout to the viral load. These approaches enable rapid viral diagnostics within 30 min of leaf harvest, making them valuable for onsite field applications. Timely identification of diseases associated with pathogens is crucial for effective treatment; yet developing rapid, specific, sensitive, and cost-effective diagnostic technologies remains challenging. The current gold standard, PCR technology, has drawbacks such as lengthy operational cycles, high costs, and demanding requirements. Here we update the technical advancements of CRISPR-Cas in viral detection, providing insights into future developments, versatile applications, and potential clinical translation. There is a need for approaches enabling field plant viral nucleic acid detection with high sensitivity, specificity, affordability, and portability. Despite challenges, CRISPR-Cas-mediated pathogen diagnostic solutions hold robust capabilities, paving the way for ideal diagnostic tools. Alternative applications in virus research are also explored, acknowledging the technology's limitations and challenges.