A novel indirect ELISA for serodiagnosis of mucormycosis using antigens from Rhizopus arrhizus.

BACKGROUND: Due to a delay in diagnosis by conventional techniques and high mortality, the development of a standardised and rapid non-culture-based technique is an unmet need in pulmonary, gastrointestinal, and disseminated forms of mucormycosis. Though limited studies have been conducted for molecular diagnosis, there are no established serologic tests for this highly fatal infection. OBJECTIVE: To develop and evaluate an indirect in-house enzyme-linked immunosorbent assay (ELISA) utilising antigens of Rhizopus arrhizus for detecting anti-Rhizopus antibodies (IgG and IgM) in sera of patients with mucormycosis. METHODS: We extracted both secretory and mycelial Rhizopus antigens using standardised protocols. Bradford assay was used for protein quantification. We then standardised an indirect ELISA using R. arrhizus mycelial and secretory antigens (10.0 µg/mL in bicarbonate buffer pH 9.2) for detecting anti-Rhizopus IgG and IgM antibodies in patient sera. We included patients with mucormycosis, other fungal infections, and healthy controls. Antibody index value (E-value) was calculated for each patient sample. RESULTS: Asparagine broth culture filtrate utilising 85% ammonium sulphate salt fractionation and mycelial homogenate grown in yeast extract peptone dextrose (YPD) broth precipitated with trichloroacetic acid (TCA) yielded a large amount of good-quality protein for the assay. We included 55 patients with mucormycosis (rhino-orbito-cerebral mucormycosis [ROCM, n = 39], pulmonary [n = 15], gastrointestinal [n = 1]), 24 with other fungal infections (probable aspergillosis [n = 14], candidiasis [n = 10]), and healthy controls (n = 16). The sensitivity of the antibody test for diagnosing mucormycosis ranged from 83.6-92.7% for IgG and 72.7-87.3% for IgM, with a specificity of 91.7-92.5% for IgG and 80-82.5% for IgM. The sera from patients with other fungal infections and healthy individuals did not show significant cross-reactivity. CONCLUSION: The detection of anti-Rhizopus IgG antibody performed significantly better in comparison to IgM-based ELISA for diagnosing both ROCM (sensitivity of 84.6% vs. 69.2%) and pulmonary cases (86.6% vs. 80.0%). More extensive studies are required to confirm our findings.

In vitro and in vivo evaluation of DNase I in reinstating antibiotic efficacy against Klebsiella pneumoniae biofilms.

Klebsiella pneumoniae is an opportunistic pathogen associated with biofilm-based infections, which are intrinsically antibiotic resistant. Extracellular DNA plays a crucial role in biofilm formation and self-defence, with nucleases being proposed as promising agents for biofilm disruption. This study evaluated the in vitro and in vivo efficacy of DNase I in improving the activity of cefotaxime, amikacin, and ciprofloxacin against K. pneumoniae biofilms. K. pneumoniae ATCC 700603 and a clinical isolate from catheter-related bloodstream infection were cultured for biofilm formation on microtiter plates, and the antibiofilm activity of the antibiotics (0.03-64 mg/L), with or without bovine pancreatic DNase I (1-32 mg/L) was determined by XTT dye reduction test and viable counting. The effect of ciprofloxacin (2 mg/L) and DNase I (16 mg/L) was further evaluated in vitro on 1-cm-long silicon catheter segments, and in a mouse model of subcutaneous catheter-associated infection. Combination with DNase I did not improve the biofilm-preventive capacity of the three antibiotics or the biofilm-eradicating capacity of cefotaxime and amikacin. The biofilm-eradicating capacity of ciprofloxacin was increased by 8-fold and 4-fold in K. pneumoniae ATCC 700603 and clinical isolate, respectively, with DNase I. The combination therapy caused 99% reduction in biofilm biomass in the mouse model.

Meta-Analysis Identifies BDNF and Novel Common Genes Differently Altered in Cross-Species Models of Rett Syndrome.

Rett syndrome (RTT) is a rare disorder and one of the most abundant causes of intellectual disabilities in females. Single mutations in the gene coding for methyl-CpG-binding protein 2 (MeCP2) are responsible for the disorder. MeCP2 regulates gene expression as a transcriptional regulator as well as through epigenetic imprinting and chromatin condensation. Consequently, numerous biological pathways on multiple levels are influenced. However, the exact molecular pathways from genotype to phenotype are currently not fully elucidated. Treatment of RTT is purely symptomatic as no curative options for RTT have yet to reach the clinic. The paucity of this is mainly due to an incomplete understanding of the underlying pathophysiology of the disorder with no clinically useful common disease drivers, biomarkers, or therapeutic targets being identified. With the premise of identifying universal and robust disease drivers and therapeutic targets, here, we interrogated a range of RTT transcriptomic studies spanning different species, models, and MECP2 mutations. A meta-analysis using RNA sequencing data from brains of RTT mouse models, human post-mortem brain tissue, and patient-derived induced pluripotent stem cell (iPSC) neurons was performed using weighted gene correlation network analysis (WGCNA). This study identified a module of genes common to all datasets with the following ten hub genes driving the expression: ATRX, ADCY7, ADCY9, SOD1, CACNA1A, PLCG1, CCT5, RPS9, BDNF, and MECP2. Here, we discuss the potential benefits of these genes as therapeutic targets.

One-year continuous abstinence rate for smoking cessation via telephonic counselling: The Indian scenario.

Tobacco control methods differ by country, with telephonic counseling being one of them. The effectiveness of telephone counseling in smoking cessation has been discussed on several occasions. India's tobacco problem is more complex than that of any other country in the world. To begin with, tobacco is consumed in a variety of ways, and India is a large multilingual country with remarkable cultural diversity. In India, the National Tobacco Quitline Service (NTQLS) is a government-run program. Its data from May 2016 to May 2021 were analyzed retrospectively in this cross-sectional study to determine the prevalence and pattern of tobacco use in India, as well as the abstinence rate for smoking cessation. A total of 4,611,866 calls were received by the Interactive Voice Response system (IVR). The number of calls increased from 600 to 5400 per day after the toll-free number was printed on all tobacco products. Smokeless tobacco use was discovered to be more prevalent, with males significantly more likely to use both smoking and smokeless tobacco. At one month and one year after quitting, 33.42% and 21.9%, respectively, remained tobacco-free. The study emphasizes the efficacy of behavioral counseling in increasing abstinence rates. The printing of a toll-free number on tobacco products is an effective strategy for expanding the operation of quit lines. Despite the challenges of cultural diversity and complex tobacco use, India's quit line service has been able to provide counseling to callers with prolonged abstinence and quit rates comparable to the various quit lines around the world.

The Indigenous Volatile Inhibitor 2-Methyl-2-butene Impacts Biofilm Formation and Interspecies Interaction of the Pathogenic Mucorale Rhizopus arrhizus.

2-Methyl-2-butene has recently been reported to be a quorum-based volatile self-inhibitor of spore germination and growth in pathogenic Mucorale Rhizopus arrhizus. The present study aimed to elucidate if this compound can influence R. arrhizus biofilm formation and interspecies interaction. The compound was found to significantly decrease R. arrhizus biofilm formation (p < 0.001), with nearly 25% and 50% lesser biomass in the biofilms cultured with exposure to 4 and 32 µg/ml of 2-methyl-2-butene, respectively. The growth of pre-formed biofilms was also impacted, albeit to a lesser extent. Additionally, 2-methyl-2-butene was found to self-limit R. arrhizus growth during interspecies interaction with Staphylococcus aureus and was detected at a substantially greater concentration in the headspace of co-cultures (2338.75 µg/ml) compared with monocultures (69.52 µg/ml). Some of the C5 derivatives of this compound (3-methyl-1-butanol, 2-methyl-2-butanol, and 3-methyl-1-butyne) were also observed to partially mimic its action, such as inhibition of spore germination, but did not impact R. arrhizus biofilm formation. Finally, the treated R. arrhizus displayed changes in fungal morphology suggestive of cytoskeletal alterations, such as filopodia formation, blebs, increased longitudinal folds and/or corrugations, and finger-like and sheet-like surface protrusions, depending upon the concentration of the compound(s) and the planktonic or biofilm growth mode.

Nasal Microbiota Imbalance as a Contributory Link in the Emergence of COVID-19 Associated Mucormycosis.

COVID-19 associated mucormycosis (CAM) is being reported at an elevated frequency and has been declared an ongoing epidemic in India. A huge diabetic population, inappropriate corticosteroid usage and environmental mucoralean spore count, along with COVID-19 associated glycemic imbalance, hypoxemia, increased iron levels, vascular endothelial injury, as well as the immunosuppressive impact are being considered as important risk factors for CAM. The present viewpoint aims to discuss the plausible role of another important facet, the nasal microbiota imbalance, in the emergence of mucormycosis under the prevailing COVID-19 pandemic conditions.

Rapid and Simple Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) Method for Simultaneous Quantifications of Triazole Antifungals in Human Serum.

PURPOSE: To develop and validate a one-step, rapid and simple reversed-phase high-performance liquid chromatography (HPLC)-based protocol for the simultaneous measurement of voriconazole (VCZ), posaconazole (POSA), itraconazole (ITC) in serum/plasma. METHODS: Calibration standards (CS) and quality control samples were prepared in drug-free serum by spiking with the triazoles at different concentrations. HPLC was performed with C18 column, isocratic mobile phase after extraction with cold acetonitrile. The standardized method was tested in 2693 patients' serum/plasma samples. RESULTS: Linearity of CS for ITC, VCZ and POSA was proportional to the nominal concentration (correlation coefficient > 0.999). Limit of detection (mg/L) for ITC, VCZ and POSA was 0.25, 0.25 and 0.125, respectively. The lower limit of quantification (mg/L) for ITC, VCZ and POSA was 0.5, 0.5 and 0.25, respectively. Precision and accuracy were in acceptable range with 100% average percentage recovery. No interferences from endogenous substances and other antimicrobial compounds were noted. In clinical samples, the therapeutic range achieved for VCZ was 39.9%. Whereas, 61.1% and 44% of samples with ITC and POSA, respectively, were in the sub-therapeutic range. CONCLUSION: We developed a rapid and simple HPLC method to quantify common triazoles in a single chromatographic run allowing simultaneous measurement of different antifungals in a small volume of serum/plasma. Thus, therapeutic drug monitoring requests can be processed in one run without changing the protocol parameters, column or column conditioning thereby improving turnaround time.

Microfilaria in achylous hematuria: Can it imitate urolithiasis?

Wuchereria bancrofti is the major species resulting in filarial cases in India. Filariasis may present with various clinical presentations: asymptomatic, microfilaremia, lymphedema, acute adenolymphangitis and chronic lymphatic disease. Microfilariae of W. bancrofti have been detected in various clinical samples, but incidental detection of microfilaria in achylous urine is a rare finding with unexplained pathology. We report a case of microfilaria in achylous urine of a 30-year-old female who presented with pain abdomen and ureteric calculus on ultrasonography.

Volatile self-inhibitor of spore germination in pathogenic Mucorale Rhizopus arrhizus.

Rhizopus arrhizus is a common pathogenic Mucoralean mold that exists as a saprophyte, and is disseminated through sporangiospores, which germinate to form mycelia under suitable environmental or infection settings. Such morphological transitions are often mediated by self-produced effector molecules in a density-dependent fashion. This study aimed to elucidate if a quorum-dependent, cell-density-driven phenomenon exists in R. arrhizus, and identify the molecule(s) involved. The germination of R. arrhizus was observed to be reliant on the seeding density, with nearly 71% and 47% germination in Sabouraud dextrose and glucose asparagine media respectively at 1 × 105-1 × 106 spores/mL, and only 10% and 1% germination respectively with 1 × 108 spores/mL. The late-growth-stage supernatant also hindered the spore germination and liquid-culture biomass in a dose-dependent way. These effects were being mediated by a volatile inhibitor present in the headspace and supernatant of R. arrhizus cultures, identified as 2-methyl-2-butene by gas chromatography and electron ionization-quadrupole mass spectrometry. The compound was present in a density-dependent manner and considerably impaired fungal germ-tube emergence and elongation during germination. Spore swelling remained unaffected. Multiple thin protrusions comprising of F-actin and microtubules were seen emanating from the treated cells, suggestive of filopodia-like and cytoneme-like extensions. The same compound was also detected in Rhizomucor pusillus.

Facially Amphiphilic Cholic Acid-Lysine Conjugates as Promising Antimicrobials.

The emergence of multidrug-resistant microbes is a significant health concern posing a constant need for new antimicrobials. Membrane-targeting antibiotics are promising candidates with reduced ability of microbes to develop resistance. In the present investigation, the principal reason behind choosing cholic acid as the crucial scaffold lies in the fact that it has a facially amphiphilic nature, which provides ample opportunity to refine the amphiphilicity by linking the amino acid lysine. A total of 16 novel amphipathic cholic acid derivatives were synthesized by sequentially linking lysine to C3-ß-amino cholic acid methyl ester to maintain the hydrophobic/hydrophilic balance, which could be the essential requirement for the antimicrobial activity. Among the synthesized conjugates, a series with fluorenyl-9-methoxycarbonyl moiety attached to cholic acid via lysine linker showed promising antimicrobial activity against Staphylococcus aureus, Escherichia coli, and Candida albicans. A pronounced effect of increase in lysine residues was noted on the observed activity. The lead compounds were found to be active against drug-resistant bacterial and fungal clinical isolates and also improved the efficacy of antifungal agents amphotericin B and voriconazole. Membrane-permeability studies demonstrated the ability of these compounds to induce membrane damage in the tested microbes. The active conjugates did not show any hemolytic activity and were also found to be nontoxic to the normal cells as well as the examined cancer cell lines. The observed antimicrobial activity was attributed to the facial amphiphilic conformations, hydrophobic/hydrophilic balance, and the overall charge on the molecules.

Antagonistic Interaction of Staphylococcus aureus and Staphylococcus epidermidis with Rhizopus arrhizus Mediated by Phenol Soluble Modulins and Organic Acids.

Rhizopus arrhizus (R. arrhizus) is a common causative agent of mucormycosis that usually enters the human body through the respiratory tract and skin. Both these sites harbor staphylococci as a part of the normal microflora, indicating the possibility of interspecies interactions. We aimed to elucidate this interaction and identify the molecular mechanisms involved. Both Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) substantially hindered R. arrhizus radial growth, spore germination, and liquid culture biomass. Secreted components in the stationary-phase supernatant were responsible for this activity. The active components, based on molecular weight-based fractionation, mass spectrometry, and ion exclusion chromatography, were identified as a truncated version of phenol soluble modulin α2 (Δ1Δ2PSMα2) and PSMα3 in S. aureus, PSMδ in S. epidermidis, and organic acids in both the species. Exposure to the phenol soluble modulins (PSMs) extensively damaged the fungal spores and pre-existing hyphae, leading to bleb formation, shriveling, hyphal shrinkage, and cell distortion.

Medically important interactions of staphylococci with pathogenic fungi.

Staphylococci are common inhabitants at several human body sites and are also implicated in infections either as primary or opportunistic pathogens. These bacteria can thus both contribute to the host defense being a part of the commensalistic microbiota or synergize with the other microbes during the infection process. Among fungi, staphylococci interact synergistically with Candida spp. and Aspergillus fumigatus, and antagonistically with Cryptococcus neoformans and Trichosporon asahii. These interactions are highly dynamic and are orchestrated by a multitude of microbial and host factors. During such cross-talks, staphylococci can modulate the virulence, immune response or drug resistance of the coexisting microbe(s), thereby influencing the infection course, disease severity, treatment strategy and the clinical outcome.

Biofilm formation by Candida auris isolated from colonising sites and candidemia cases.

BACKGROUND: Candida auris, an emerging nosocomial pathogen, exhibits phenotypic variation. Non-aggregating C. auris isolates display greater biofilm-forming capacity and virulence than aggregate-forming isolates. Most of the studies till date have focused on clinical isolates. The biofilm-forming capacity of colonising isolates remains uninvestigated. OBJECTIVES: The present study aimed to elucidate the biofilm-forming capacity of the colonising isolates of C. auris, correlate it with their aggregation behaviour and antifungal susceptibility, and compare it with that of the isolates from blood-stream infection. METHODS: Colonising and clinical (candidemia) isolates of C. auris were screened for aggregation behaviour, biofilm-forming capacity and antifungal susceptibility testing. Aggregation behaviour was assessed microscopically. Biofilm-forming capacity was determined on 96-well flat-bottomed microtitre plates. Antifungal susceptibility testing was performed by broth microdilution assay. RESULTS: Aggregative and non-aggregative phenotypes were found to be predominantly associated with colonising and clinical isolates, respectively, with the former ones being stronger biofilm producers in the colonising group. Non-aggregative isolates in the colonising group showed lower susceptibility to amphotericin B and fluconazole than aggregative isolates. In contrast, no association was noted between biofilm formation, aggregation behaviour and antifungal susceptibility amongst the clinical isolates. CONCLUSION: Biofilm formation is a strain-dependent trait in C. auris, strongly associated with the type and phenotypic behaviour of the isolates. Colonising isolates of this fungus were found to be predominantly aggregative in nature, with a higher biofilm-forming capacity than non-aggregative ones.

Use of papaya leaf extract in neonatal thrombocytopenia.

Thrombocytopenia is a common condition in neonates. Neonatal thrombocytopenia can cause serious complications such as intraventricular hemorrhage. Papaya leaf extract may be a safe and effective therapy in persistent refractory neonatal thrombocytopenia. Papaya leaf extract was effective for improving platelet count in our baby with persistent thrombocytopenia. However, with no previous evidence of its use in neonates, its use is still experimental and should only be considered in refractory cases after team meeting and detailed discussion with family.

Health benefits of resveratrol: Evidence from clinical studies.

Resveratrol is a polyphenolic nutraceutical that exhibits pleiotropic activities in human subjects. The efficacy, safety, and pharmacokinetics of resveratrol have been documented in over 244 clinical trials, with an additional 27 clinical trials currently ongoing. Resveretrol is reported to potentially improve the therapeutic outcome in patients suffering from diabetes mellitus, obesity, colorectal cancer, breast cancer, multiple myeloma, metabolic syndrome, hypertension, Alzheimer's disease, stroke, cardiovascular diseases, kidney diseases, inflammatory diseases, and rhinopharyngitis. The polyphenol is reported to be safe at doses up to 5 g/d, when used either alone or as a combination therapy. The molecular basis for the pleiotropic activities of resveratrol are based on its ability to modulate multiple cell signaling molecules such as cytokines, caspases, matrix metalloproteinases, Wnt, nuclear factor-κB, Notch, 5'-AMP-activated protein kinase, intercellular adhesion molecule, vascular cell adhesion molecule, sirtuin type 1, peroxisome proliferator-activated receptor-γ coactivator 1α, insulin-like growth factor 1, insulin-like growth factor-binding protein 3, Ras association domain family 1α, pAkt, vascular endothelial growth factor, cyclooxygenase 2, nuclear factor erythroid 2 like 2, and Kelch-like ECH-associated protein 1. Although the clinical utility of resveratrol is well documented, the rapid metabolism and poor bioavailability have limited its therapeutic use. In this regard, the recently produced micronized resveratrol formulation called SRT501, shows promise. This review discusses the currently available clinical data on resveratrol in the prevention, management, and treatment of various diseases and disorders. Based on the current evidence, the potential utility of this molecule in the clinic is discussed.

Bile Acid Oligomers and Their Combination with Antibiotics To Combat Bacterial Infections.

The ever-growing risk of bacterial resistance is a critical concern. Among the various antimicrobial resistant bacterial strains, methicillin and vancomycin resistant Staphylococcus aureus are among the most dreadful, causing serious complications. On the basis of the hypothesis that microbes have reduced ability to develop resistance against membrane targeting antibiotics, bile acid oligomers having unique facially amphiphilic topologies were designed and synthesized. The oligomers with specific linkers exhibited potent and selective antibacterial activity against Gram-positive bacteria. The lead compounds also improved the efficacy of a range of known antibiotics belonging to different classes when tested in combination. The active dimers were found to be effective against antibiotic-resistant clinical isolates of S. aureus, including multidrug resistant isolates. A significant inhibitory activity against S. aureus biofilm, a highly drug-resistant bacterial phenotype often unresponsive to antibiotic therapy, was also noticed. No adverse effects were observed by these dimers in a cell viability assay against HEK293 cells.

Role of biofilm morphology, matrix content and surface hydrophobicity in the biofilm-forming capacity of various Candida species.

The present study aimed to evaluate the role of biofilm morphology, matrix content and surface hydrophobicity in the biofilm-forming capacity of Candida albicans and non-albicans Candida (NAC) spp. Biofilm formation was determined by microtitre plate assay and bright-field and scanning electron microscopy. The matrix carbohydrates, proteins and e-DNA were quantified by phenol-sulfuric acid, bicinchoninic acid and UV spectroscopy, respectively. Specific glycosyl residues were detected by dot blot. The cell-surface hydrophobicity was determined by hydrocarbon adhesion assay. Candida tropicalis was found to exhibit the highest adherence to polystyrene. It formed dense biofilms with extensive pseudohyphae and hyphal elements, high hydrophobicity and the greatest amount of matrix carbohydrates, proteins and e-DNA. C. albicans displayed higher adherence and a complex biofilm morphology with larger aggregates than Candida parapsilosis and Candida krusei, but had lower matrix content and hydrophobicity. Thus, the combinatorial effect of increased filamentation, maximum matrix content and high hydrophobicity contributes to the enhanced biofilm-forming capacity of C. tropicalis.