Utility of serum Galactomannan in diagnosing COVID-19 patients with suspected IPA: an observational study in resource limited settings.

OBJECTIVE: To study the utility of Galactomannan (GM) antigen as a screening marker for diagnosing invasive pulmonary aspergillosis (IPA) in coronavirus disease 2019 (COVID-19) patients. PATIENTS AND METHODS: The serum samples from patients with severe COVID-19 diseases admitted to the Critical Care Unit were collected on the 5th day of admission for GM screening. The samples were analysed by enzyme linked immune sorbent assay (ELISA) and GM index of more than 1 was considered as positive. All GM positive patients were serially followed until discharge or death. RESULTS: The GM was raised in serum of 12 out of 38 patients, indicating an incidence of possible COVID-19 associated IPA (CAPA) in 31.57% of patients. The median age of these CAPA patients was 56.5 years, males were significantly more affected than females. The inflammatory marker serum ferritin was raised in all 12 patients (median value of 713.74 ng/ml), while IL-6 was raised in 9 patients (median value of 54.13 ng/ml). None of these patients received antifungals. Their median length of hospital stay was 20 days (IQR: 12, 34 days). All these patients succumbed to the illness. CONCLUSIONS: The serum GM appears to be sensitive diagnostic tool to identify early IPA in COVID-19 patients and pre-emptive antifungal therapy could play a role in salvaging these patients.

Terbinafine resistance in dermatophytes: Time to revisit alternate antifungal therapy.

Resistant superficial dermatophytic infections of the skin and its appendages have emerged as a major health problem in India. Mutations in Squalene epoxidase gene have led to increasing incidence of resistance to terbinafine in dermatophytic isolates. We examined six patients with recalcitrant dermatophytosis attending Dermatology OPD at a tertiary care hospital and demonstrated terbinafine resistance by molecular method. Immediate hyperitivity (IH) reaction to Trichophytin antigen was highlighted in these patients. The patients were treated with alternate antifungals after demonstration of resistance to terbinafine based on the antifungal susceptibility testing (AFST). On follow up the patients responded well to the substitute but the duration of therapy had to be prolonged beyond six weeks.

Candida auris in critically ill patients: Emerging threat in intensive care unit of hospitals.

Candida auris has emerged as the multi-drug resistant non-albican candida species in critically ill patients admitted to intensive care units (ICU) causing bloodstream and other infections. It has often been misidentified as Candida famata, Candida sake, Rhodotorula glutinis, or Saccharomyces cerevisiae and Candida haemulonii by automated identification systems. We, in this study, discuss three patients who acquired Candida auris infection after 15 to 20days of their stay in medical ICU. Medical equipment, use of multiple antibiotics, and poor hand hygiene are the most probable predisposing factors attributing to its colonization at multiple anatomical sites leading to bloodstream infection. Candida auris might substantially contaminate the environment of colonized or infected patients making its eradication difficult. Patient screening for Candida auris, especially during prolonged ICU stays, along with strict infection prevention and control strategies needs to be adopted to break its persistence.

Development of methodologies for the regioselective synthesis of four series of regioisomer isoxazoles from ß-enamino diketones.

Four methodologies are reported for the regioselective synthesis of four series of regioisomer isoxazoles from cyclocondensation of ß-enamino diketones and hydroxylamine hydrochloride. Regiochemical control was achieved by varying reaction conditions and substrate structure. The mild reaction conditions used to access 4,5-disubstituted, 3,4-disubtituted, and 3,4,5-trisubstituted regioisomer isoxazoles, as well as the pharmacological and synthetic potential of the products, make these novel methodologies very powerful.

Development of a real-time loop-mediated isothermal amplification assay for detection of Burkholderia mallei.

Burkholderia mallei is the aetiological agent of glanders, a highly contagious and re-emerging zoonotic disease. Early diagnosis of glanders is critically important to ensure timely treatment with appropriate antibiotics in humans, and to prevent spread of infection in animals. Molecular detection of B. mallei has always been troublesome because of its genetic similarity with Burkholderia pseudomallei, the causative agent of melioidosis. In present investigation, a set of six B. mallei-specific primers were designed and a simple, rapid, specific and sensitive real-time loop-mediated isothermal amplification (LAMP) assay was developed for detection of B. mallei. The LAMP assay could detect as low as 1 pg of B. mallei genomic DNA and 5.5 × 103  CFU/ml of B. mallei in spiked human blood. The assay was highly specific for B. mallei as it did not cross-react with other bacterial strains used in the study. The established LAMP assay is field adaptable and can be a better and viable alternative to PCR-based techniques for detection of B. mallei in glanders endemic areas with resource-limited settings.

Challenges in culture-negative cases of Madurella mycetomatis: A case report re-accentuating PCR as an essential diagnostic tool.

Identification of dematiaceous fungi responsible for black-grain mycetoma has remained cumbersome and time consuming for years leading to delayed diagnosis and thereby increased agony to patients. Moreover, difficult morphology of some of these fungi demanding enough expertise for species identification in addition to culture-negativity has often led to misdiagnosis and hence inapt treatment to the patients. We report the identification of Madurella mycetomatis from culture-negative black granules discharged from foot nodular lesions of a 27 years old male using PCR followed by sequencing of the internal transcribed spacer region. The patient's lesions were successfully treated using a combination of itraconazole (200mg) and terbinafine (250mg), confirming our diagnosis. Our case study proves the clinical value of PCR as the best, rapid and accurate diagnostic method for the identification of Madurella mycetomatis and related fungi, particularly in culture-negative cases.

Synthesis and evaluation against Leishmania amazonensis of novel pyrazolo[3,4-d]pyridazinone-N-acylhydrazone-(bi)thiophene hybrids.

A new series of pyrazolo[3,4-d]pyridazin-7-one derivatives were synthesised and evaluated for their in vitro antileishmanial activity against Leishmania amazonensis promastigote and axenic amastigote forms. The results showed that the pyrazolo[3,4-d]-pyridazin-7-one-N-acylhydrazone-(bi)thiophene hybrids 5b, 6b and 6d exhibit better antileishmanial activity with IC50 84.96, 3.63 and 10.79 µM, against the promastigote form and IC50 32.71, 2.32 and >100 µM against the axenic amastigote form, respectively. The active compounds had their cytotoxicity tested against macrophages and fibroblast cells with a higher selectivity index than 10 for compounds 6b and 6d. Molecular docking studies were performed for all active compounds using the enzyme trypanothione reductase (TR) to investigate a possible action mechanism. The results suggested that active compounds had interactions with the residues of amino acids Gly 13, Thr 51, Thr 160, Gly 161, Tyr 198, Arg 287, Asp 327, Thr 335, which may inhibit the enzyme TR.

Evaluation of Parmotrema reticulatum Taylor for Antibacterial and Antiinflammatory Activities.

Lichens produce variety of secondary metabolites including depsides, depsidones and dibenzofurans having multifunctional activity in response to external environmental condition. Present study provides evidence for in vitro antibacterial and in vivo antiinflammatory activity of acetone and ethanol extracts of Parmotrema reticulatum. In vitro antibacterial activity was investigated against gram positive and gram negative bacteria. Cotton pellet-induced granuloma, xylene-induced ear swelling, carragennan-induced edema, histamine-induced and carboxymethylcellulose sodium-induced leukocyte emigration in mice models were used to quantify the antiinflammatory activity. Acetone and ethanol extracts were showed antibacterial activity against Bacillus subtilis (minimal inhibitory concentration: 100 and 150 µg/ml) and Staphylococcus aureus (minimal inhibitory concentration: 100 and 200 µg/ml), Escherichia coli (minimal inhibitory concentration: 200 and 250 µg/ml), and Pseudomonasa eruginosa (minimal inhibitory concentration: 200 and 300 µg/ml). Acetone extract was inhibited edema significantly at 200 mg/kg with xylene, cotton pellet, carragennan and histamine induced edema in vivo models. Ethanol extract was found effective at dose of 300 mg/kg with all in vivo antiinflammatory models. The results showed significant (P<0.01) antiinflammatory effects at 200 and 300 mg/kg dose of acetone and ethanol extracts, respectively, which can be concluded that significant activity may be due to presence of flavanoids in ethanol extract and usnic acid in acetone extract.

NMR spectroscopy and theoretical calculations in the conformational analysis of 1-methylpyrrolidin-2-one 3-halo-derivatives.

This study reports the results of ab initio and density functional theory (DFT) electronic structure calculations as well as (3)J(HH) experimental and calculated coupling constant data obtained in the investigation of the conformational equilibrium of 3-halo-derivatives of 1-methylpyrrolidin-2-one. The five-membered ring assumes an envelope conformation owing to the plane of formation of the O═C-N-R bond, with C4 forming the "envelope lid". When the conformation changes, the "lid" alternates between positions above and below the amide plane. The α-carbonyl halogen assumes two positions: a pseudo-axial and a pseudo-equatorial. In the gaseous phase, the calculations indicate that the pseudo-axial conformer is more stable and preferable going down the halogen family. Natural bond orbital analysis showed that electronic delocalization is significant only for the iodo derivative. In the other derivatives, the electrostatic repulsion between oxygen and the halogen determines the conformational equilibrium. When the solvated molecule was taken into account, the pseudo-equatorial conformer population increased with the relative permittivity of the solvent. This variation was strong in the fluoro derivative, and the preference was inverted. In the chlorine derivative, the two populations became closer in methanol and acetonitrile. In the bromine and iodine derivatives, the percentage of pseudo-equatorial conformer increased only slightly owing to the dipole moment of the conformation: the pseudo-equatorial conformation has a greater dipole moment and thus is stable in media with high relative permittivity.

Subcutaneous mycoses caused by Rhytidhysteron species in an immunocompetent patient.

Subcutaneous mycoses are chronic fungal infections of the skin and subcutaneous tissues caused by variety of fungal agents and usually occur following trauma with vegetative matter. We report a case of subcutaneous mycoses caused by rare fungus belonging to the genus Rhytidhysteron, in an immunocompetent male who presented with a subcutaneous nodule on left foot. This unusual species was identified and confirmed by molecular methods.

12-lipoxygenase activity plays an important role in PAR4 and GPVI-mediated platelet reactivity.

Following initial platelet activation, arachidonic acid is metabolised by cyclooxygenase-1 and 12-lipoxygenase (12-LOX). While the role of 12-LOX in the platelet is not well defined, recent evidence suggests that it may be important for regulation of platelet activity and is agonist-specific in the manner in which it regulates platelet function. Using small molecule inhibitors selective for 12-LOX and 12-LOX-deficient mice, the role of 12-LOX in regulation of human platelet activation and thrombosis was investigated. Pharmacologically inhibiting 12-LOX resulted in attenuation of platelet aggregation, selective inhibition of dense versus alpha granule secretion, and inhibition of platelet adhesion under flow for PAR4 and collagen. Additionally, 12-LOX-deficient mice showed attenuated integrin activity to PAR4-AP and convulxin compared to wild-type mice. Finally, platelet activation by PARs was shown to be differentially dependent on COX-1 and 12-LOX with PAR1 relying on COX-1 oxidation of arachidonic acid while PAR4 being more dependent on 12-LOX for normal platelet function. These studies demonstrate an important role for 12-LOX in regulating platelet activation and thrombosis. Furthermore, the data presented here provide a basis for potentially targeting 12-LOX as a means to attenuate unwanted platelet activation and clot formation.

Oxadiazole 2-oxides are toxic to the human hookworm, Ancylostoma ceylanicum, however glutathione reductase is not the primary target.

Hookworm disease, characterized by severe anemia and cognitive and growth delays, currently affects an estimated 740 million people worldwide. Despite the prevalence of this parasitic disease, few effective drug therapies are in use today, and the heavy reliance upon benzimidazoles highlights the need for the development of novel chemotherapies. Recent work with the trematode parasite Schistosoma mansoni has identified oxadiazole 2-oxides as effective antischistosomal compounds that function by targeting and inhibiting the antioxidant enzyme, thioredoxin glutathione reductase. In this study, a related enzyme, glutathione reductase, from the human hookworm Ancylostoma ceylanicum was identified and characterized, and its in vitro activity in the presence of the oxadiazole 2-oxides was analyzed. Ex vivo worm killing assays were also conducted to establish the relationship between a given compound's effect upon worm survival and inhibition of recombinant glutathione reductase (rAceGR). Finally, the in vivo anthelminthic efficacy of furoxan (Fx) was assessed in the hamster model of hookworm infection. The predicted amino acid sequence of AceGR contained a prototypical glutathione reductase active site sequence, but no thioredoxin reductase consensus sequences, suggesting that the glutathione and thioredoxin pathways of A. ceylanicum are distinct. Although ten of the forty-two oxadiazole 2-oxides tested inhibited rAceGR activity by at least fifty percent, and fifteen compounds were toxic to parasites ex vivo, little overlap existed between these two results. We therefore suggest that AceGR is not the primary target of the oxadiazole 2-oxides in effecting parasite death. Lastly, oral treatment of A. ceylanicuminfected hamsters with furoxan resulted in significantly improved weight gains and reduced intestinal worm burdens compared to vehicle treated controls, supporting continued development of this molecule as a novel anthelminthic.

Real-time polymerase chain reaction assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder.

Real-time polymerase chain reaction (real-time PCR) is a laboratory technique based on PCR. This technique is able to detect sequence-specific PCR products as they accumulate in "real time" during the PCR amplification, and also to quantify the number of substrates present in the initial PCR mixture before amplification begins. In the present study, real-time PCR assay was employed for rapid and real-time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 5 to 10(7) spores. DNA was isolated from spiked soil and talcum powder, using PBS containing 1 % Triton-X-100, followed by heat treatment. The isolated DNA was used as template for real-time PCR and PCR. Real-time PCR amplification was obtained in 60 min under the annealing condition at 60°C by employing primers targeting the pag gene of B. anthracis. In the present study, the detection limit of real-time PCR assay in soil was 10(3) spores and 10(2) spores in talcum powder, respectively, whereas PCR could detect 10(4) spores in soil and 10(3) spores in talcum powder, respectively.

Protective effect of curcumin, silymarin and N-acetylcysteine on antitubercular drug-induced hepatotoxicity assessed in an in vitro model.

Tuberculosis (TB) is highly endemic in India. The first-line anti-TB therapy (ATT) involving isoniazid (INH), rifampicin and pyrazinamide causes hepatotoxicity in approximately 11.5% of Indian patients. Studies have shown that ATT-induced hepatotoxicity is primarily due to oxidative stress caused by the drugs and metabolites. Herbal drugs with antioxidative properties have been tested in animal studies and clinical trials for the management of hepatotoxicity. The objective of this study was to investigate the role of curcumin (CUR), silymarin (SILY) and N-acetylcysteine (N-ACET) on hepatotoxicity by ATT drugs using an in vitro model of human hepatocellular carcinoma cell line (HepG2). HepG2 cells were treated with ATT drugs alone or along with CUR, SILY or N-ACET for a 48-h duration. The cells were monitored for viability, morphology, respiring mitochondria and cell cycle. Our results suggest that the presence of hepatoprotective drugs during treatment of HepG2 cells with ATT drugs lowers the hepatotoxic effect of the latter. This is observed in terms of (a) increased cell viability, (b) healthy-looking cell morphology as revealed by phase contrast microscopy, (c) active respiring cells as observed with confocal microscopy upon staining with a mitochondrial membrane-specific dye, MitoTracker(®) Red, and reduction in the sub-G(1) peak in cell cycle analysis by flow cytometry. Our results suggest that these hepatoprotective drugs need to be further explored as potential adjuvant therapy along with ATT drugs.

Comparative genetic analysis of trichome-less and normal pod genotypes of Mucuna pruriens (Fabaceae).

Velvet bean (Mucuna pruriens) seeds contain the catecholic amino acid L-DoPA (L-3,4-dihydroxyphenylalanine), which is a neurotransmitter precursor and used for the treatment of Parkinson's disease and mental disorders. The great demand for L-DoPA is largely met by the pharmaceutical industry through extraction of the compound from wild populations of this plant; commercial exploitation of this compound is hampered because of its limited availability. The trichomes present on the pods can cause severe itching, blisters and dermatitis, discouraging cultivation. We screened genetic stocks of velvet bean for the trichome-less trait, along with high seed yield and L-DoPA content. The highest yielding trichome-less elite strain was selected and indentified on the basis of a PCR-based DNA fingerprinting method (RAPD), using deca-nucleotide primers. A genetic similarity index matrix was obtained through multivariant analysis using Nei and Li's coefficient. The similarity coefficients were used to generate a tree for cluster analysis using the UPGMA method. Analysis of amplification spectra of 408 bands obtained with 56 primers allowed us to distinguish a trichome-less elite strain of M. pruriens.

Design and synthesis of immunoconjugates and development of competition inhibition enzyme-linked immunosorbent assay (CIEIA) for the detection of O-isopropyl methylphosphonofluoridate (sarin): an organophosphorous toxicant.

Three haptens of the organophosphorus (OP) toxicant 'sarin' having different spacer arm were designed and synthesized. Haptens were conjugated with BSA (bovine serum albumin) and ovalbumin (OVA) for raising antibody and coating antigen. High antibody titer with higher specificity was obtained from 4-(4-(isopropoxy(methyl)phosphoryloxy)phenylamino)-4-oxobutanoic acid (hapten B) having reasonable long spacer arm. For the standard curve, an IC(50) (inhibitory concentration) of free antigen was found to be 0.415 µg mL(-1) on the basis of indirect competitive ELISA. The study revealed that heterology in competition inhibition enzyme immunoassay (CIEIA) produced remarkable improvement in the sensitivity and specificity of the assay. Under the optimized conditions, the quantitative working range was found to be 0.19-1.56 µg mL(-1) with a limit of detection (LOD) of 0.05 µg mL(-1). The antibodies showed negligible cross reactivity (CR) with other OP toxicants and pesticides, which makes the assay suitable for the selective detection of sarin.

Photocatalytic inactivation of spores of Bacillus anthracis using titania nanomaterials.

Studies on photocatalytic inactivation of spores of Bacillus anthracis have been carried out using nanosized titania materials and UVA light or sun light. Results demonstrated pseudo first order behaviour of spore inactivation kinetics. The value of kinetic rate constant increased from 0.4h(-1) to 1.4h(-1) indicating photocatalysis facilitated by addition of nanosized titania. Nanosized titania exhibited superior inactivation kinetics on par with large sized titania. The value of kinetic rate constant increased from 0.02 h(-1) to 0.26 h(-1) on reduction of size from 1000 nm to 16 nm depicting the enhanced rate of inactivation of Bacillus anthracis Sterne spores on the decrease of particle size.

Real-time loop-mediated isothermal amplification assay for rapid and sensitive detection of anthrax spores in spiked soil and talcum powder.

Loop-mediated isothermal amplification (LAMP) assay is a powerful and innovative gene amplification technique that specifically amplifies the target gene under isothermal conditions with a high degree of sensitivity, rapidity and specificity. The major advantage of the LAMP assay is monitoring of amplified products without the requirement of any sophisticated equipment. In the present study a real time LAMP assay was employed for rapid and real time detection of Bacillus anthracis spores spiked in 0.1 g of soil and talcum powder ranging from 2 to 10(7) spores. DNA was isolated from spiked soil and talcum powder using PBS containing 1% Triton X-100, and heat treatment. Isolated DNA was used as template for LAMP and PCR. LAMP amplification was obtained in 60 min under isothermal condition at 63°C by employing a set of six primers targeting the pag gene of B. anthracis. The detection limit of LAMP assay in soil and talcum powder was found to be as low as 5 spores, compared to 10(3) spores and 10(4) spores by PCR in talcum powder and soil, respectively. The findings suggest that LAMP is a more rapid and sensitive assay than PCR for detecting anthrax spores, additionally the methodology to prepare DNA from spiked samples is simple, rapid and cost effective.

Evaluation of PCR, DNA hybridization and immunomagnetic separation - PCR for detection of Burkholderia mallei in artificially inoculated environmental samples.

Glanders is highly contagious disease of equines, caused by Burkholderia mallei. The disease though rare, can be transmitted to humans. Here, we report a strategy for rapid detection of B. mallei from environmental samples. Different bacteriological media were evaluated and brain heart infusion broth medium with selective supplements (BHIB-SS) of penicillin (200 U/ml) and crystal violet (1:10,00000) was found to support the maximum growth of B. mallei even in the presence of other bacteria like Escherichia coli and Staphylococcus aureus. A polymerase chain reaction (PCR) and a DNA hybridization method was standardized for 823 bp specific dNA sequence of B. mallei. To enable the quicker and direct enrichment of B. mallei bacteria from environmental samples, an immunomagnetic separation (IMS) method was also standardized. Water, husk, grass and gram samples were artificially contaminated by B. mallei bacteria and after enrichment of B. mallei in BHIB-SS, detection was carried out by PCR and DNA hybridization. PCR was found to be a better method of the two with a detection limit of 10(4)-10(6) CFU/ml (6 h enrichment in BHIB-SS) in water and other particulate matrices. Detection by PCR in the above samples without enrichment in BHIBSS was carried out following IMS where the detection limit was about 1-2 log higher than PCR following enrichment in BHIB-SS. We recommend PCR for 823 bp for detection of B. mallei from environmental samples either following enrichment in BHIB-SS or IMS. IMS-PCR method may be preferred in situations where numbers of B. mallei bacteria are expected to be high and results are required in short time.

Medicinal mushrooms: Towards a new horizon.

The arising awareness about functional food has created a boom in this new millennium. Mushrooms are widely consumed by the people due to their nutritive and medicinal properties. Belonging to taxonomic category of basidiomycetes or ascomycetes, these mushrooms possess antioxidant and antimicrobial properties. They are also one of the richest source of anticancer and immunomodulating agents. Thus these novel myochemicals from these mushrooms are the wave of future.