Mucormycosis: Cytomorphological Spectrum in Fine-Needle Aspiration Cytology.

Background: Mucormycosis is a fungal infection that can affect multiple organs. The role of fine-needle aspiration cytology (FNAC) in its diagnosis is not well documented. Aim: The objective of this study was to describe the detailed cytomorphologic features of mucormycosis on FNAC samples. Materials and Methods: A retrospective analysis of all cases diagnosed as mucormycosis on FNAC between January 2014 and July 2021 was performed for detailed cytomorphological evaluation and correlation to clinical data and microbiological studies wherever available. FNA was computed tomography-guided (n = 38), ultrasonography-guided (n = 31) or palpation-guided (n = 12), and slides were reviewed in two cases. Results: A total of 83 cases of mucormycosis were evaluated. An immunocompromised setting was observed in 48 cases. The most common site of FNA was the lung (n = 57) followed by liver, soft tissue, palate, mediastinum, orbital/ocular region, and lymph node. Isolated renal involvement, a unique feature, was seen in seven cases. The aspirates were necrotic to pus-like or blood-mixed particulate. Broad, nonseptate, foldable, ribbon-like fungal hyphae showing right-angled branching were seen. The tissue reaction was predominantly necro-inflammatory (n = 36), bland necrotic (n = 22), mixed inflammatory (n = 16), suppurative (n = 5), necrotizing granulomatous (n = 3), and granulomatous (n = 1). Immunocompromised patients showed mixed inflammatory responses more frequently. Fungal culture was positive for Rhizopus species in 2/13 cases and molecular testing in two additional cases corresponding to Rhizopus and Syncephalastrum spp. Conclusion: FNA provides quick and conclusive diagnosis of mucormycosis from varied anatomic sites enabling prompt institution of therapy. The tissue response is variable and to some extent dependent on the immune status of the patient.

Immune and metabolic perturbations in COVID-19-associated pulmonary mucormycosis: A transcriptome analysis of innate immune cells.

BACKGROUND AND OBJECTIVES: The mechanisms underlying COVID-19-associated pulmonary mucormycosis (CAPM) remain unclear. We use a transcriptomic analysis of the innate immune cells to investigate the host immune and metabolic response pathways in patients with CAPM. PATIENTS AND METHODS: We enrolled subjects with CAPM (n = 5), pulmonary mucormycosis (PM) without COVID-19 (n = 5), COVID-19 (without mucormycosis, n = 5), healthy controls (n = 5) without comorbid illness and negative for SARS-CoV-2. Peripheral blood samples from cases were collected before initiating antifungal therapy, and neutrophils and monocytes were isolated. RNA sequencing was performed using Illumina HiSeqX from monocytes and neutrophils. Raw reads were aligned with HISAT-2 pipeline and DESeq2 was used for differential gene expression. Gene ontology (GO) and metabolic pathway analysis were performed using Shiny GO application and R packages (ggplot2, Pathview). RESULTS: The derangement of core immune and metabolic responses in CAPM patients was noted. Pattern recognition receptors, dectin-2, MCL, FcRγ receptors and CLEC-2, were upregulated, but signalling pathways such as JAK-STAT, IL-17 and CARD-9 were downregulated; mTOR and MAP-kinase signalling were elevated in monocytes from CAPM patients. The complement receptors, NETosis, and pro-inflammatory responses, such as S100A8/A9, lipocalin and MMP9, were elevated. The major metabolic pathways of glucose metabolism-glycolysis/gluconeogenesis, pentose phosphate pathway, HIF signalling and iron metabolism-ferroptosis were also upregulated in CAPM. CONCLUSIONS: We identified significant alterations in the metabolic pathways possibly leading to cellular iron overload and a hyperglycaemic state. Immune responses revealed altered recognition, signalling, effector functions and a pro-inflammatory state in monocytes and neutrophils from CAPM patients.

Molecular cartography uncovers evolutionary and microenvironmental dynamics in sporadic colorectal tumors.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Analysis of spatial multi-omic data from 31 human colorectal specimens enabled phylogeographic mapping of tumor evolution that revealed individualized progression trajectories and accompanying microenvironmental and clonal alterations. Phylogeographic mapping ordered genetic events, classified tumors by their evolutionary dynamics, and placed clonal regions along global pseudotemporal progression trajectories encompassing the chromosomal instability (CIN+) and hypermutated (HM) pathways. Integrated single-cell and spatial transcriptomic data revealed recurring epithelial programs and infiltrating immune states along progression pseudotime. We discovered an immune exclusion signature (IEX), consisting of extracellular matrix regulators DDR1, TGFBI, PAK4, and DPEP1, that charts with CIN+ tumor progression, is associated with reduced cytotoxic cell infiltration, and shows prognostic value in independent cohorts. This spatial multi-omic atlas provides insights into colorectal tumor-microenvironment co-evolution, serving as a resource for stratification and targeted treatments.

Cladosporium halotolerans: Exploring an Unheeded Human Pathogen.

BACKGROUND: Cladosporium halotolerans is a saprobic fungus, rarely implicated in human infections. The identification is challenging due to non-specific phenotypic features. OBJECTIVE: To decipher clinical spectrum, microbiological and susceptibility profile of clinical and environmental isolates of Cladosporium halotolerans. METHOD: All the isolates identified as Cladosporium halotolerans deposited in National Culture Collection for Pathogenic Fungi (NCCPF), Postgraduate Institute of Medical Education and Research, Chandigarh, India were revived. Phenotypic and molecular characterization targeting internal transcribed spacer (ITS) region of ribosomal DNA, large subunit of ribosomal DNA (LSU; NL1 and NL4), actin (ACT) and beta-tubulin (TUB) was done. Scanning electron microscopy (SEM) was performed to determine any phenotypic variations. Antifungal susceptibility testing (AFST) was carried out for eight antifungal agents as per CLSI M38 Ed3 guidelines. We also performed systematic literature review of all the cases of Cladosporium halotolerans reported till date. RESULTS: A total of four isolates (clinical, n = 3; soil, n = 1) identified as Cladosporium halotolerans were included in the study. The clinical sites were skin, maxillary tissue and nail. All patients were apparently immunocompetent, and history of trauma was recorded in one patient. All patients improved on antifungal therapy. The cultures revealed growth of black mycelial fungus and microscopic examination demonstrated dematiaceous septate hyphae with erect conidiophores and conidia in branched acropetal chains. Based on molecular methods, all the four isolates were identified as C. halotolerans. SEM revealed no variation in length and width of the conidia, conidiophores, ramoconidium and hyphae among the isolates. All molecular targets, such as ITS region, LSU (partially sequenced), ACT and TUB were able to differentiate the isolates. Minimum inhibitory concentrations for antifungals were: triazoles (0.12-2 µg/ml), amphotericin B (4 µg/ml) and echinocandins (2-8 µg/ml). CONCLUSION: We report role of the rarely isolated dematiaceous fungus, C. halotolerans, in causing human infections. The study emphasizes the role of molecular methods in precisely identifying these species. Triazoles are more active against these black fungi compared to polyenes or echinocandins.

Reflecting on changes in the drinking and irrigation water quality of rivers Beas, Satluj and confluence waters.

Prevention and control of water pollution for maintaining and restoring the wholesomeness of rivers are unavoidable. The current water quality approach of designated best use has some limitations such as it is non-integrative and inflexible with regard to the consideration of variables and does not provide a separate rating scale for a given designated use. We thus used water quality index approach proposed by the Canadian Council of Ministers of the Environment (CCME WQI) to evaluate and develop a separate rating system for drinking and irrigation purposes of rivers Beas, Satluj and their confluence water of the Indian Punjab using information collected over 4 years (2016 to 2019). River Beas exhibited better water quality compared to river Satluj for irrigation as well as for drinking. The overall drinking water quality index (DWQI) for Beas was marginal (45.5), whereas it was poor for Satluj (37.7) and confluence waters (40.1). The spatial variation in DWQI was greater for Satluj compared to Beas and confluence waters reflecting the effect of dumping of untreated industrial and domestic waste waters. Variables such as Total coliform (T. coli), dissolved oxygen (DO), turbidity and biological oxygen demand (BOD) contributed to the deterioration of DWQI. The irrigation water quality index (IWQI) was good for Beas (86), marginal for Satluj (60.1) and fair for confluence waters (71.2). Faecal coliform (F. coli), Kelly ratio (KR) and %Na contributed to the deterioration of IWQI. Calcium-magnesium-bicarbonate (Ca-Mg-HCO3) was the dominant water type in Beas and confluence waters, whereas for Satluj, in addition to Ca-Mg-HCO3, sodium-potassium-chloride-sulphate and mixed water types were also prevalent. The river waters witnessed salinity hazard but did not pose sodicity hazard except at a few locations of Satluj. The study indicates the need to take location specific measures for improving river water quality for drinking as well as irrigation purposes. The current status of water quality calls for an urgent need to formulate stringent policy regulations to maintain the surface water quality.

A Specialized Epithelial Cell Type Regulating Mucosal Immunity and Driving Human Crohn's Disease.

Crohn's disease (CD) is a complex chronic inflammatory disorder that may affect any part of gastrointestinal tract with extra-intestinal manifestations and associated immune dysregulation. To characterize heterogeneity in CD, we profiled single-cell transcriptomics of 170 samples from 65 CD patients and 18 non-inflammatory bowel disease (IBD) controls in both the terminal ileum (TI) and ascending colon (AC). Analysis of 202,359 cells identified a novel epithelial cell type in both TI and AC, featuring high expression of LCN2, NOS2, and DUOX2, and thus is named LND. LND cells, confirmed by high-resolution in-situ RNA imaging, were rarely found in non-IBD controls, but expanded significantly in active CD. Compared to other epithelial cells, genes defining LND cells were enriched in antimicrobial response and immunoregulation. Moreover, multiplexed protein imaging demonstrated that LND cell abundance was associated with immune infiltration. Cross-talk between LND and immune cells was explored by ligand-receptor interactions and further evidenced by their spatial colocalization. LND cells showed significant enrichment of expression specificity of IBD/CD susceptibility genes, revealing its role in immunopathogenesis of CD. Investigating lineage relationships of epithelial cells detected two LND cell subpopulations with different origins and developmental potential, early and late LND. The ratio of the late to early LND cells was related to anti-TNF response. These findings emphasize the pathogenic role of the specialized LND cell type in both Crohn's ileitis and Crohn's colitis and identify novel biomarkers associated with disease activity and treatment response.

Scedosporium Infection in Recipients of Kidney Transplants from Deceased Near-Drowning Donor.

Scedosporium aurianticum infection developed in 2 recipients of kidney transplants in India, acquired from the same deceased near-drowning donor. Given the substantial risk for death associated with Scedosporium infection among solid-organ transplant recipients, safety protocols for organ transplantation from nearly drowned donors should be thoroughly revaluated and refined.

GammaGateR: semi-automated marker gating for single-cell multiplexed imaging.

Motivation: Multiplexed immunofluorescence (mIF) is an emerging assay for multichannel protein imaging that can decipher cell-level spatial features in tissues. However, existing automated cell phenotyping methods, such as clustering, face challenges in achieving consistency across experiments and often require subjective evaluation. As a result, mIF analyses often revert to marker gating based on manual thresholding of raw imaging data. Results: To address the need for an evaluable semi-automated algorithm, we developed GammaGateR, an R package for interactive marker gating designed specifically for segmented cell-level data from mIF images. Based on a novel closed-form gamma mixture model, GammaGateR provides estimates of marker-positive cell proportions and soft clustering of marker-positive cells. The model incorporates user-specified constraints that provide a consistent but slide-specific model fit. We compared GammaGateR against the newest unsupervised approach for annotating mIF data, employing two colon datasets and one ovarian cancer dataset for the evaluation. We showed that GammaGateR produces highly similar results to a silver standard established through manual annotation. Furthermore, we demonstrated its effectiveness in identifying biological signals, achieved by mapping known spatial interactions between CD68 and MUC5AC cells in the colon and by accurately predicting survival in ovarian cancer patients using the phenotype probabilities as input for machine learning methods. GammaGateR is a highly efficient tool that can improve the replicability of marker gating results, while reducing the time of manual segmentation. Availability and Implementation: The R package is available at https://github.com/JiangmeiRubyXiong/GammaGateR.

Comparative Evaluation of Low-level Diode Laser and Electrosurgical Pulpotomy in Primary Molars.

Aim: Considering the advantages of the nonpharmacotherapeutic techniques over the disadvantages of the pharmacotherapeutic agents, there is a need to evaluate clinically as well as radiographically various nonpharmacotherapeutic techniques to fortify them as replacements to the traditional pulpotomy. This study was conducted to evaluate and compare the clinical and radiographic outcomes of low-level diode laser (LLDL) and electrosurgical pulpotomy in primary molars. Materials and methods: Seventy primary molars were allocated to group I (n = 35) and group II (n = 35), which underwent LLDL pulpotomy and electrosurgical pulpotomy, respectively. Clinical and radiographic analysis of all the teeth in the two groups was performed at 1, 3, 6, 9, and 12 months, respectively. Pearson's Chi-squared test was utilized to evaluate the success of both treatment procedures (p < 0.05). Results: During the 12-month follow-up period, the differences between the groups pertaining to clinical and radiographical evaluation were statistically nonsignificant, although enhanced results were seen in the laser pulpotomy group. There was no significant difference in the success rate observed between the LLDL pulpotomy and electrosurgical pulpotomy group at the end of the 12-month follow-up period. Conclusion: The two pulpotomy techniques were found to be successful enough in strengthening the concept of a potent and safe nonpharmacotherapeutic approach in the management of pulpally involved primary molars. Clinical significance: This study further establishes nonpharmacological pulpotomy techniques as a favorable alternative to traditional pulpotomy methods. How to cite this article: Roy S, Garg N, Pathivada L, et al. Comparative Evaluation of Low-level Diode Laser and Electrosurgical Pulpotomy in Primary Molars. Int J Clin Pediatr Dent 2023;16(4):612-618.

Recent advances in the synthesis and applications of single-atom nanozymes in food safety monitoring.

Nanozymes are synthetic compounds with enzyme-like tunable catalytic properties. The success of nanozymes for catalytic applications can be attributed to their small dimensions, cost-effective synthesis, appreciable stability, and scalability to molecular dimensions. The emergence of single atom nanozymes (SANzymes) has opened up new possibilities in bioanalytical applications. In this regard, this review outlines enzyme-mimicking features of SANzymes for food safety applications in relation to the key variables controlling their catalytic performance. The discussion is extended further to cover the applications of SANzymes for the monitoring of various compounds/biomaterials of significance with respect to food safety (e.g., pesticides, veterinary drug residues, foodborne pathogenic bacteria, mycotoxins/bacterial endotoxin, antioxidant residues, hydrogen peroxide residues, and heavy metal ions). Furthermore, the performance of SANzymes is evaluated in terms of various performance metrics such as limit of detection (LOD), linear dynamic range, and figure of merit (FoM). The challenges and future road map for the applications of SANzymes are also addressed along with their upscaling in the area of food safety.

Cytology of Rhytidhysteron rufulum: An emerging cause of phaeohyphomycosis.

Fine-needle aspiration cytology (FNAC) is often the first-line investigation for detection of any fungal infection. Rhytidhysteron rufulum is an emerging dematiaceous fungus detected as a human pathogen. FNAC combined with molecular techniques helps in the detection of rare fungal species, especially in cases of non-sporulating fungi. We describe the cytomorphologic features of this species in a 62-year immunocompetent male who presented with a localised subcutaneous infection. Molecular studies helped in the final diagnosis.

Correlation of Serum Vitamin D with Crestal Bone Level in Dental Implant Patients Using CBCT: A Clinical Retrospective Study.

AIM: The purpose of this study is to correlate the role of serum vitamin D levels associated with crestal bone in dental implant patients using cone-beam computed tomography (CBCT). MATERIALS AND METHODS: This retrospective study enrolled patients among whom implants were placed after a detailed planning using CBCT and the delayed loading protocol was followed. After 3 months and 6 months of loading, CBCT evaluations were carried out, serum vitamin D levels were also tested at the end of 6 months of loading. A total of 30 patients were recruited with 15 patients in each group based on normal and deficient levels of vitamin D to correlate with crestal bone levels using CBCT. RESULTS: Clinically acceptable crestal bone loss (CBL) was visible with all the implants at different time intervals. Statistical analysis was done for intergroup and intragroup comparisons which showed significant p-value (< 0.05) for CBL at the time of loading, at 3 months, and at 6 months follow-up for both normal and deficient serum values. In the deficient group, the mean value at baseline was 9.69 mm ± 1.10 and the CBL at 6 months follow-up was 8.80 mm ± 1.10 whereas for the normal group at baseline, the mean was 9.08 mm ± 1.21 and at 6 months follow-up was 8.12 mm ± 1.25 which showed meaningful difference. CONCLUSION: There is a positive correlation seen between CBL on CBCT and vitamin D serum levels. The success of the implant is significantly affected by vitamin D as it regulates the bone physiology and has systemic effects on accelerating bone formation around titanium implants. CLINICAL SIGNIFICANCE: Vitamin D is essential in maintaining the balance of bone minerals and assists to preserve the crestal bone level making the implant treatment more predictable and successful.

Application of Novel Short Tandem Repeat Typing for Wickerhamomyces anomalus Reveals Simultaneous Outbreaks within a Single Hospital.

Wickerhamomyces anomalus, previously known as Candida pelliculosa, occasionally causes candidemia in humans, primarily infecting neonates, and infants. The mortality rate of these invasive infections is high, and isolates with a reduced susceptibility to fluconazole have been reported. W. anomalus outbreaks are regularly reported in healthcare facilities, especially in neonatal intensive care units (NICUs). In order to rapidly genotype isolates with a high-resolution, we developed and applied a short tandem repeat (STR) typing scheme for W. anomalus. Six STR markers were selected and amplified in two multiplex PCRs, M3 and M6, respectively. In total, 90 W. anomalus isolates were typed, leading to the identification of 38 different genotypes. Four large clusters were found, unveiling simultaneous outbreak events spread across multiple units within the same hospital. STR typing results of 11 isolates were compared to whole-genome sequencing (WGS) single nucleotide polymorphism (SNP) calling, and the identified genotypic relationships were highly concordant. We performed antifungal susceptibility testing of these isolates, and a reduced susceptibility to fluconazole was found for two (2.3%) isolates. ERG11 genes of these two isolates were examined using WGS data, which revealed a novel I469L substitution in one isolate. By constructing a homology model for W. anomalus ERG11p, the substitution was found in close proximity to the fluconazole binding site. In summary, we showed multiple W. anomalus outbreak events by applying a novel STR genotyping scheme.

Cannabinoid receptor type 1 regulates sequential stages of migration and morphogenesis of neural crest cells and derivatives in chicken and frog embryos.

The main cannabinoid receptor CB1R first shows expression during early neurula stage in chicken (Gallus gallus) embryos, and at early tailbud stage in the frog (Xenopus laevis) embryos. This raises the question of whether CB1R regulates similar or distinct processes during the embryonic development of these two species. Here, we examined whether CB1R influences the migration and morphogenesis of neural crest cells and derivatives in both chicken and frog embryos. Early neurula stage chicken embryos were exposed to arachidonyl-2'-chloroethylamide (ACEA; a CB1R agonist), N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; a CB1R inverse agonist) or Blebbistatin (nonmuscle Myosin II inhibitor) in ovo and examined during migration of neural crest cells and at condensing cranial ganglia stage. Early tailbud stage frog embryos were bathed in ACEA, AM251 or Blebbistatin, and analyzed at late tailbud stage for changes in craniofacial and eye morphogenesis, and in patterning and morphology of melanophores (neural crest-derived pigment cells). In chicken embryos exposed to ACEA and Myosin II inhibitor, cranial neural crest cells migrated erratically from the neural tube, and the right, but not the left, ophthalmic nerve of the trigeminal ganglia was affected in ACEA- and AM251-treated embryos. In frog embryos with inactivation or activation of CB1R, or inhibition of Myosin II, the craniofacial and eye regions were smaller and/or less developed, and the melanophores overlying the posterior midbrain were more dense, and stellate in morphology, than the same tissues and cells in control embryos. This data suggests that despite differences in the time of onset of expression, normal activity of CB1R is required for sequential steps in migration and morphogenesis of neural crest cells and derivatives in both chicken and frog embryos. In addition, CB1R may signal through Myosin II to regulate migration and morphogenesis of neural crest cells and derivatives in chicken and frog embryos.

Clinical and Mycologic Characteristics of Emerging Mucormycosis Agent Rhizopus homothallicus.

We retrospectively reviewed consecutive cases of mucormycosis reported from a tertiary-care center in India to determine the clinical and mycologic characteristics of emerging Rhizopus homothallicus fungus. The objectives were ascertaining the proportion of R. homothallicus infection and the 30-day mortality rate in rhino-orbital mucormycosis attributable to R. homothallicus compared with R. arrhizus. R. homothallicus accounted for 43 (6.8%) of the 631 cases of mucormycosis. R. homothallicus infection was independently associated with better survival (odds ratio [OR] 0.08 [95% CI 0.02-0.36]; p = 0.001) than for R. arrhizus infection (4/41 [9.8%] vs. 104/266 [39.1%]) after adjusting for age, intracranial involvement, and surgery. We also performed antifungal-susceptibility testing, which indicated a low range of MICs for R. homothallicus against the commonly used antifungals (amphotericin B [0.03-16], itraconazole [0.03-16], posaconazole [0.03-8], and isavuconazole [0.03-16]). 18S gene sequencing and amplified length polymorphism analysis revealed distinct clustering of R. homothallicus.

The significance of APACHE II as a predictor of mortality in paraquat poisoning: A systematic review and meta-analysis.

The Acute Physiology and Chronic Health Evaluation II (APACHE II) scoring system is utilised as a prognostic method in paraquat poisoning; however, current evidence shows ambiguity. Although some studies have shown APACHE II to be a superior tool, others have reported it inferior to other prognostic markers, such as lactate, severity index of paraquat poisoning and urine paraquat concentration. Hence, to address this ambiguity, we conducted a systematic review and meta-analysis to analyse prognostic accuracy of APACHE II score in predicting mortality in paraquat poisoning. We included twenty studies with 2524 paraquat poisoned patients in the systematic review, after a comprehensive literature search in databases PubMed, Embase, Web of Science, Scopus and Cochrane Library, from which 16 studies were included in the meta-analysis. The survivors of paraquat poisoning were found to have significantly lower APACHE II scores (Mean Difference (MD): -5.76; 95% CI: -7.93 to -3.60 p < 0.0001; n = 16 studies) compared to non-survivors. The pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio (DOR) for APACHE II score <9 was found to be 74%, 68%, 2.58, 0.38 and 7.10, respectively (n = 5 studies). The area under the curve (AUC) of the bivariate summary receiver operating characteristic (SROC) curve was found to be 0.80. The pooled sensitivity, specificity, PLR, NLR and DOR for APACHE II score ≥9 was found to be 73%, 86%, 4.69, 0.33 and 16.42, respectively (n = 9 studies). The AUC of the SROC curve was found to be 0.89. Pairwise AUC comparison of APACHE II with other prognostic markers showed serum presepsin to have a significantly better discriminatory ability than APACHE II. Through the findings of this study, we conclude that APACHE II was found to be a good indicator of death in paraquat poisoning patients. However, higher APACHE II scores (≥9) depicted greater specificity in predicting mortality in paraquat poisoning. Thus, APACHE II can be used as a practical tool in the hand of physicians to prognose patients with paraquat poisoning to aid clinical decisions.