Structural insights into loss of function of a pore forming toxin and its role in pneumococcal adaptation to an intracellular lifestyle.

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane ß-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche.

Psychiatric Sequelae and COVID Experiences of Post COVID -19 Recovered Resident Doctors and Interns of a Tertiary General Hospital in Mumbai.

Background: The wide and profound psychological impact of the COVID pandemic on people has been demonstrated by several studies. A survey from the Indian Medical Association (IMA) showed that at least 500 doctors have been infected across Maharashtra, with Mumbai doctors making it to the top of the list. The most infected are the resident doctors working in government hospitals. A few doctors also have lost their lives while working in this pandemic. The delicate balance between duty, altruism and fear for oneself and others often causes conflict and feelings of helplessness and dissonance in many health care personnel with a fear to resume back the duty, especially after being a COVID victim. There have been hardly any studies yet, related to understanding the emotional and general well being of COVID-19 positive health care workers who have recovered and resumed their duties. Aims and Objectives: To study in post COVID-19 recovered resident doctors and interns the presence of anxiety and depressive symptoms, sleep changes, general well-being, perceptions of their COVID experience and association of general wellbeing with anxiety, depression and sleep changes. Methods: The study was conducted after institutional ethics committee permission and online informed consent from the participants which included medical interns and resident doctors of our institute. A Google form with questions and scales pertaining to the aims of the study (Patient Health Questionnaire, Zung Self Rating Anxiety Scale, Warwick Edinburgh Mental Well Being Scale and Insomnia Symptom Questionnaire) was sent on the Google link to the 150 interns and resident doctors via whatsapp who had recovered from COVID 19 infection. Their details were taken from hospital database with Dean permission. Results: 79 responded to the link with the mean duration of COVID infection being 16.1 ± 8.59 days. 64 participants were hospitalized and 18 participants had lung parenchymal involvement. 28 participants had depressive symptoms on the patient health questionnaire. Only 4 participants experienced anxiety as per Zung self rating anxiety scale. 69 participants had greater well being with scores higher than 40 on the Warwick Edinburgh mental well being scale. 8 participants had sleep related issues affecting their work. A negative correlation of general well being was seen with depression, anxiety and insomnia which was highly significant. The most distressing of COVID experiences were being isolated in a room, transmitting disease to near and dear ones and possibility of a serious complication. Re infection, workload followed by use of PPE was the major concern.Many described their COVID experience as boring, depressing with feelings of loneliness. Conclusions: Our study is the first of its kind to evaluate the psychiatric sequelae and COVID experiences of post COVID-19 recovered resident doctors and interns. Psychosocial and institutional support will definitely help in improving the post COVID sequelae in the resident doctors and interns.

Circulating ensembles of tumor-associated cells: A redoubtable new systemic hallmark of cancer.

Circulating ensembles of tumor-associated cells (C-ETACs) which comprise tumor emboli, immune cells and fibroblasts pose well-recognized risks of thrombosis and aggressive metastasis. However, the detection, prevalence and characterization of C-ETACs have been impaired due to methodological difficulties. Our findings show extensive pan-cancer prevalence of C-ETACs on a hitherto unreported scale in cancer patients and virtual undetectability in asymptomatic individuals. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of 16,134 subjects including 5,509 patients with epithelial malignancies in various organs and 10,625 asymptomatic individuals with age related higher cancer risk. PBMCs were treated with stabilizing reagents to protect and harvest apoptosis-resistant C-ETACs, which are defined as cell clusters comprising at least three EpCAM+ and CK+ cells irrespective of leucocyte common antigen (CD45) status. All asymptomatic individuals underwent screening investigations for malignancy including PAP smear, mammography, low-dose computed tomography, evaluation of cancer antigen 125, cancer antigen 19-9, alpha fetoprotein, carcinoembryonic antigen, prostate specific antigen (PSA) levels and clinical examination to identify healthy individuals with no indication of cancer. C-ETACs were detected in 4,944 (89.8%, 95% CI: 89.0-90.7%) out of 5,509 cases of cancer. C-ETACs were detected in 255 (3%, 95% CI: 2.7-3.4%) of the 8,493 individuals with no abnormal findings in screening. C-ETACs were detected in 137 (6.4%, 95% CI: 5.4-7.4%) of the 2,132 asymptomatic individuals with abnormal results in one or more screening tests. Our study shows that heterotypic C-ETACs are ubiquitous in epithelial cancers irrespective of radiological, metastatic or therapy status. C-ETACs thus qualify to be a systemic hallmark of cancer.

Analyzing Cell-free Genomic DNA in Spent Culture Media: Noninvasive Insight into the Blastocysts.

A commonly accepted standard protocol for noninvasive techniques for the genetic evaluation of an embryo remains elusive due to inconclusiveness regarding the volume of spent media to be acquired and the possibility of acquiring the same for subsequent analysis. Single embryo culture is imperative for standardizing noninvasive preimplantation testing using cell-free DNA (cf-DNA) released by individual developing embryos. This study aims to compare the development dynamics of single-drop embryonic culture against with group embryonic culture to establish a standardized protocol for noninvasive Preimplantation Genetic Testing (PGT) in bovine. A total of 239 cumulus-oocyte complexes were aspirated and subjected to in vitro maturation and fertilization. Among these, 120 embryos of day 3 were transferred to single-drop culture until the blastocyst stage. The single-drop culture drops were prepared using microdrops of 30 µL. At the blastocyst stage, spent media from all single-drop embryos were utilized for extracting cell-free genomic DNA to standardize the protocol. The blastocyst rate indicates no significant difference between the two culture methods, suggesting that single-drop culture is suitable for the process. Additionally, the extracted spent media yielded sufficient quantities of cf-DNA, supporting its potential use for PGT ( p < 0.05). These findings support the hypothesis that single-drop embryo culture is a viable method for cf-DNA extraction and confirm the potential of using DNA fragments from spent media as a reliable source for noninvasive PGT.

Brolucizumab-associated intraocular inflammation in Indian patients by VRSI study group.

CONTEXT: Concerns about brolucizumab's (Pagenax®) association with intraocular inflammation (IOI) limit its use despite its cost-effectiveness and efficacy. This multicentric study analyzes IOI incidence across 21 tertiary eyecare centers in India since its introduction in October 2020. PURPOSE: To determine the real-world incidence rate of IOI in Indian patients secondary to intravitreal brolucizumab across 21 tertiary eye care centers in India. SETTINGS AND DESIGN: Retrospective multicentric, survey-based study. METHODS: Data including number of patients treated, clinical indications, side effects encountered, and IOI case details was collected via Google Forms in 21 Indian tertiary eye care centers since October 2020. Mean, median, frequency, and standard deviation were calculated for statistical analysis. RESULTS: All centers used pro re nata protocol for brolucizumab injections with a minimum injection interval of 8 weeks. The incidence of IOI was 0.79% (21 events out of 2655 eyes). Treatment indications included idiopathic polypoidal choroidal vasculopathy, neovascular age-related macular degeneration, diabetic macular edema, and off-label uses. IOI was experienced after the first injection (57%) in majority of cases with a median onset of 14 days (range: 1-65 days). IOI was mild in 28.5%, moderate in 33%, and severe in 38% of cases. Eighteen out of 21 IOI eyes recovered preinjection best corrected visual acuity or better. CONCLUSIONS: Our study found a lower IOI incidence (0.79%) with brolucizumab (Pagenax) in Indian patients compared to previously reported literature. IOI events were mostly mild to moderate, and post-treatment, most patients improved or maintained BCVA. Larger prospective multicentric studies with PRN dosing protocol are needed to confirm these findings.

Comparison of clinical examination, doppler and fistulogram for arterio-venous fistula surveillance in detecting secondary failure.

BACKGROUND: Arteriovenous Fistula (AVF) surveillance is required to detect early dysfunction (thrombosis, stenosis) and its timely correction prolongs access-patency. Clinical examination (CE) and doppler have been used as screening/surveillance of AVF, for early detection of AVF dysfunction. Since there was inadequate evidence for KDOQI to make recommendations on AVF surveillance and on secondary failure rate. We compared CE, doppler and fistulogram as surveillance modalities in detecting a secondary failure in matured AVF. METHODOLOGY: This prospective-observational, single-center study, was done between December 2019-April 2021. CKD stage 5 patients on dialysis/Not-on-dialysis with matured AVF were included at third month. CE, doppler (blood flow, vein diameter, depth), and fistulogram were done at third and sixth month. Secondary failure was assessed at sixth month classifying AVF to patent/functional and failed group. Diagnostic tests were performed by comparing three methods considering fistulogram as gold-standard. Residual urine output is also monitored to look for any contrast induced residual renal function loss. RESULTS: Of total 407 created AVF, 98 (24%) had primary failure. Twenty-five (6%) had surgical complications including unsuccessful AVF and aneurysm/rupture, 156 lost follow-up at third month, 104 consented patients were enrolled, 16 lost to follow-up subsequently, and 88 patients' data were analyzed at the end. At the sixth month, 76(86.4%) had patent AVF, 8 (9.1%) had secondary failure (Thrombosis-4, Central Venous Stenosis-4), and 4 (4.1%) patients expired. Considering fistulogram as a diagnostic standard, CE showed 87.5% sensitivity, and 93.4% specificity (cohen's kappa value of 0.66). Doppler had sensitivity and specificity of 87% and 96% respectively (cohen's kappa value of 0.75), Combination of clinical examination with doppler showed sensitivity and specificity of 100% and 89% respectively. CONCLUSION: Although the secondary AVF failure rate is less than the primary, CE is an important and valuable tool in the diagnosis and surveillance of AVF in detecting its dysfunction. Moreover, CE with doppler can be used as a surveillance protocol that can detect early AVF dysfunction at par with Fistulogram.

Multi-analyte liquid biopsies for molecular pathway guided personalized treatment selection in advanced refractory cancers: A clinical utility pilot study.

Purpose: The selection of safe and efficacious anticancer regimens for treatment of patients with broadly refractory metastatic cancers remains a clinical challenge. Such patients are often fatigued by toxicities of prior failed treatments and may have no further viable standard of care treatment options. Liquid Biopsy-based multi-analyte profiling in peripheral blood can identify a majority of drug targets that can guide the selection of efficacious combination regimens. Patients and methods: LIQUID IMPACT was a pilot clinical study where patients with advanced refractory cancers received combination anticancer treatment regimens based on multi-analyte liquid biopsy (MLB) profiling of circulating tumor biomarkers; this study design was based on the findings of prior feasibility analysis to determine the abundance of targetable variants in blood specimens from 1299 real-world cases of advanced refractory cancers. Results: Among the 29 patients in the intent to treat (ITT) cohort of the trial, 26 were finally evaluable as per study criteria out of whom 12 patients showed Partial Response (PR) indicating an Objective Response Rate (ORR) of 46.2% and 11 patients showed Stable Disease (SD) indicating the Disease Control Rate (DCR) to be 88.5%. The median Progression-Free Survival (mPFS) and median Overall Survival (mOS) were 4.3 months (95% CI: 3.0 - 5.6 months) and 8.8 months (95% CI: 7.0 - 10.7 months), respectively. Toxicities were manageable and there were no treatment-related deaths. Conclusion: The study findings suggest that MLB could be used to assist treatment selection in heavily pretreated patients with advanced refractory cancers.

Clinical utility of circulating tumor-associated cells to predict and monitor chemo-response in solid tumors.

PURPOSE: Selection of cytotoxic chemotherapy agents (CCA) based on pre-treatment evaluation of drug sensitivities is a desirable but unmet goal for personalized anticancer treatment strategies. Prior attempts to correlate in vitro Chemo-Response Profiles (CRP) of tumor explants or Circulating Tumor Cells (CTCs) with clinical outcomes have been largely unsuccessful. METHODS: We present results from a large cohort (n = 5090, three Arms) of patients with various solid organ tumors, where CRP of Circulating Tumor-Associated Cells (C-TACs) was determined against cancer-specific CCA panels to generate a database of 56,466 unique CRP. RESULTS: In Arm 1 (n = 230), 93.7% concordance was observed between CRP of C-TACs and concurrently obtained Tumor tissue Derived Cells (TDCs). In arm 2 (n = 2201, pretreated), resistance of C-TACs to ≥ 1 CCA was observed in 79% of cases. In a blinded subset analysis of 143 pretreated patients with radiologically ascertained disease progression, CRP of C-TACs was 87% concordant with in vivo treatment failure. In Arm 3 (n = 2734, therapy naïve), innate resistance of C-TACs to ≥ 1 CCA was observed in 61% of cases. In a blinded subset analysis of 77 therapy naïve patients, in vitro chemo-sensitivity of C-TACs was concordant with radiologically ascertained treatment response to first line CCA in 97% of cases. CONCLUSION: To our knowledge, this is the first expansive and in-depth study demonstrating that real-time CRP of C-TACs is a viable approach for non-invasive assessment of response to CCA in solid organ cancers.

Response to pazopanib-based combination regimen in a case of FGFR3 amplified gastric adenocarcinoma.

Angiogenesis inhibitors (AGI) are not presently used for the treatment of gastric cancers. This report demonstrates that angiogenesis inhibitor can be safely and effectively used in combination with cytotoxic anti-cancer agents for treatment of Gastric cancers.

Angiogenesis Inhibitors in Personalized Combination Regimens for the Treatment of Advanced Refractory Cancers.

Background: Angiogenic factors are commonly activated in solid tumors and present a viable therapeutic target. However, anticancer treatment with angiogenesis inhibitors (AGI) is limited to a few cancers, mostly as monotherapy and not selected based on molecular indications. We aimed to determine whether patient-specific combination regimens with AGI and other anticancer agents when selected based on multi-analyte tumor interrogation (ETA: Encyclopedic Tumor Analysis) can expand the scope of AGIs in advanced refractory solid organ cancers with improved treatment responses. Methods: We evaluated treatment outcomes in 60 patients with advanced, refractory solid organ cancers who received ETA-guided combination regimens of AGI with other targeted, endocrine or cytotoxic agents. Radiological evaluation of treatment response was followed by determination of Objective Response Rate (ORR), Disease Control Rate (DCR), Progression Free Survival (PFS) and Overall Survival (OS). Results: Among the 60 patients, Partial Response (PR) was observed in 28 cases (46.7%), Stable Disease (SD) was observed in 29 cases (48.3%) and Disease Progression (PD, within 60 days) was observed in 3 cases (5.0%). The ORR was 46.7% and DCR was 95.0%. At the most recent follow-up the median PFS (mPFS) was 5.0 months and median OS (mOS) was 8.9 months. There were no Grade 4 therapy related adverse events or treatment related deaths. Conclusion: ETA-guided patient-specific combination regimens with AGI and other anti-neoplastic agents, can yield improved outcomes over AGI monotherapy. Trial Registration: Details of all trials are available at WHO-ICTRP: https://apps.who.int/trialsearch/. RESILIENT ID CTRI/2018/02/011,808. LIQUID IMPACT ID CTRI/2019/02/017,548.

Liquid biopsy and multi-analyte testing guided treatment of HER2 positive periampullary adenocarcinoma with durable complete response after trastuzumab based therapy.

Periampullary adenocarcinomas are rare neoplasm that originates from the pancreatic head, the ampulla of vater, the distal bile duct or the duodenum. Surgical resection followed by adjuvant therapy is considered as the standard of care treatment for these carcinomas. Despite several advances in diagnostics and therapeutics, only 5% of these patients have an overall survival of five years or more. Currently, there is a dearth of viable therapeutic targets for this disease. The role of HER2 in cancer biology has been studied extensively in several tumour subtypes, and HER2 based targeted therapies have shown to have therapeutic benefits on different cancers. In this case report, we present a case of HER2 positive distal common bile duct carcinoma - a subtype of periampullary carcinoma with multiple relapses where multi-analyte testing with Encyclopedic Tumor Analysis (ETA) (Exacta®) identified amplification and over expression of HER2 gene which was used as a potential target to treat the patient with trastuzumab. Synchronous in vitro chemosensitivity profiling on Circulating Tumor Asscociated Cells (C-TACs) isolated from blood aided us to design the personalized chemotherapeutic regimen with cyclophosphamide and methotrexate. The combination of trastuzumab with cyclophosphamide and methotrexate yielded excellent treatment response with the patient remaining in complete response till the last follow-up. Our study suggests HER2 directed therapy as a potent pathway for treatment in the subset of HER-2 amplified distal common bile duct carcinomas.

A case report of androgen receptor inhibitor therapy in recurrent high-grade serous ovarian cancer.

Ovarian cancer is common gynaecological malignancy and a leading cause of death among women. Despite the advances in treatment strategies, majority of patients present with recurrence after first- or second-line treatment. Targeted therapy that has proven to be effective in other advanced or metastatic solid tumors have also demonstrated its efficacy in ovarian cancer. Recent studies have shown that the androgen receptor (AR) signalling is involved in pathogenicity and progression of cancer. Current observations suggest AR could be a potential target in managing the disease. In this case report we present a patient with high grade serous ovarian cancer (HGSOC) with multiple relapses with excellent disease control on AR inhibition with bicalutamide.

First look at emergency medical technician wellness in India: Application of the Maslach Burnout Inventory in an unstudied population.

INTRODUCTION: Professional wellness is critical to developing and maintaining a health care workforce. Previous work has identified burnout as a significant challenge to professional wellness facing emergency medical technicians (EMTs) in many countries worldwide. Our study fills a critical gap by assessing the prevalence of burnout among emergency medical technicians (EMTs) in India. METHODS: This was a cross-sectional survey of EMTs within the largest prehospital care organization in India. We used the Maslach Burnout Inventory (MBI) to measure wellness. All EMTs presenting for continuing medical education between July-November 2017 from the states of Gujarat, Karnataka, and Telangana were eligible. Trained, independent staff administered anonymous MBI-Medical Personnel Surveys in local languages. RESULTS: Of the 327 EMTs eligible, 314 (96%) consented to participate, and 296 (94%) surveys were scorable. The prevalence of burnout was 28.7%. Compared to EMTs in other countries, Indian EMTs had higher levels of personal accomplishment but also higher levels of emotional exhaustion and moderate levels of depersonalization. In multivariate regression, determinants of burnout included younger age, perceived lack of respect from colleagues and administrators, and a sense of physical risk. EMTs who experienced burnout were four times as likely to plan to quit their jobs within one year. CONCLUSION: This is the first assessment of burnout in EMTs in India and adds to the limited body of literature among low- and middle-income country (LMIC) prehospital providers worldwide. Burnout was strongly associated with an EMT's intention to quit within a year, with potential implications for employee turnover and healthcare workforce shortages. Burnout should be a key focus of further study and possible intervention to achieve internationally recognized targets, including Sustainable Development Goal 3C and WHO's 2030 Milestone for Human Resources.

Prosthetic management of malpositioned implant using custom cast abutment.

Two cases are reported with malpositioned implants. Both the implants were placed 6-7 months back. They had osseointegrated well with the surrounding bone. However, they presented severe facial inclination. Case I was restored with custom cast abutment with an auto polymerizing acrylic gingival veneer. Case II was restored with custom cast UCLA type plastic implant abutment. Ceramic was directly fired on the custom cast abutments. The dual treatment strategy resulted in functional and esthetic restorations despite facial malposition of the implants.