Complete genome sequencing of Kaisodi virus isolated from ticks in India belonging to Phlebovirus genus, family Phenuiviridae.

An unknown virus was repeatedly isolated from hard tick (Haemaphysalis spinigera) during a proactive arbovirus survey in ticks conducted in 1957, in India. The virus remained uncharacterized for a long time. The passages of this virus in different vertebrate and invertebrate cells along with human and monkey-derived cell culture showed no cytopathic effect. It was identified later to be a member of Kaisodi group among Phlebovirus genus in the family Phenuiviridae (Order: Bunyavirales) by serological methods. Due to its genomic diversity, sequencing of this virus was a challenge for a while. In this study, we were able to sequence the complete genome of this virus isolate using next-generation sequencing (NGS) platform. The unknown virus was identified to be Kaisodi virus (KASDV) using NGS analysis. De novo genome assembly derived three genomic segments for the KASDV which encode for RNA-dependent RNA polymerase, glycoprotein precursor, and nucleoprotein. Functional as well as conserved domains for Kaisodi serogroup viruses were predicted and compared to a known representative of the genus Phlebovirus. The phylogenetic tree revealed its closeness to Silverwater virus, of Kaisodi serogroup with nucleotide (69%, 62%, and 61%) and amino acid (52%, 51%, and 62%) identity for L, M, and S segment, respectively. The study demonstrates the presence of a conserved motif (72TRGNK76) around the RNA binding motif region in tick-borne phleboviruses. The intergenic region encompassing the S segment of Kaisodi serogroup was GC-rich whereas the other Phlebovirus had AT-rich genome. KASDV has the largest intergenic region and larger loops, suggesting stem-loops formed due to larger loops as a possible factor for instability and cause of transcription termination. This paper also describes the real-time RT-PCR and RT-PCR assays developed and used for the detection of KASDV RNA in ticks from Karnataka, Kerala and Maharashtra State, India. The KASDV positivity observed in the recently collected tick pools indicates that the KASDV, isolated from Karnataka state in 1957, is also circulating in the adjoining Kerala state. On the basis of the current study, it should be possible to develop diagnostic assays which would facilitate an in-depth field survey exploring the veterinary and medical significance of KASDV.

A closer look at the in vitro electrochemical characterisation of titanium alloys for biomedical applications using in-situ methods.

Titanium (Ti) and its alloys are widely used in several biomedical applications, particularly as permanent orthopaedic implants. Electrochemical testing provides a means to perform accelerated corrosion testing, however whilst results from polarisation testing for Ti and its alloys to date have been generally useful, they are also rather limited on the basis of several reasons. One reason is that the polarisation curves for Ti and its alloys in simulated body fluids all appear rather similar, and they do not present a classical 'breakdown' or pitting potential, making discrimination between alloys difficult. Of practical relevance however, are two key issues; (1) how do Ti alloys respond to a breakdown event? (i.e. do they readily 'repassivate'?), and, (2) what is that actual rate of Ti ion loss from exposure to physiological conditions? The answers to these questions are probed herein. Several Ti alloys of either unique composition or different fabrication method were studied, including commercially pure Ti (cp-Ti), Ti-6Al-4V, Ti-29Nb-13Ta-4.5Zr (TNTZ), selective laser melted Ti-6Al-4V, direct laser deposited cp-Ti, Ti-35Nb-15Zr, and Ti-25Nb-8Zr. Results reveal that both fabrication method and alloying influence 'repassivation' behaviour. Furthermore, atomic emission spectroelectrochemistry as applied to cp-Ti indicated actual dissolution currents of ∼2-3µA/cm-2 (i.e. ∼9µm/yr) in the range of the corrosion potential, also revealing such dissolution is persistent, even with cathodic polarisation, and definitively revealing that the presence of hydrogen peroxide and albumin activate anodic dissolution of Ti. STATEMENT OF SIGNIFICANCE: We believe the paper makes a significant and important contribution to the field of permanent implant biomaterials. Whilst we concede that the paper does not include any in vivo work, the timeliness of the work, and the completely new nature of the findings, we believe carries the impact required for Acta Biomaterialia. Key highlights include:All of the above combine to produce a manuscript that we believe has wide appeal, and can be used as both a port of reference to those working with Ti biomaterials, and also those wishing to apply useful characterisation techniques to their own work (with two very novel methods demonstrated herein, along with the unique information they provide).

Detection of multidrug-resistant tuberculosis using MGIT™(TM) and MAS-PCR in Tripura, India.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) poses a global threat that is further compounded by the human immunodeficiency virus (HIV) epidemic. OBJECTIVE: To detect MDR-TB among pulmonary TB (PTB) patients with or without HIV coinfection by isolating and identifying Mycobacterium tuberculosis from clinical samples and performing drug susceptibility testing (DST). METHODS: Sputum was collected from presumed PTB cases. Microscopic examination was performed following Ziehl-Neelsen (ZN) staining and cultured in Löwenstein-Jensen (LJ) medium. First-line anti-tuberculosis DST of the isolates was performed using MGIT™ (Mycobacterial Growth Indicator Tube) and multiplex allele-specific polymerase chain reaction (MAS-PCR). RESULTS: Of 172 study subjects, 59.3% (102/172) were smear-positive and 40.7% (70/172) were smear-negative. In the smear-positive and -negative groups, respectively 62.7% (64/102) and 8.6% (6/70) were culture-positive. DST on MGIT showed a cumulative resistance of 7.1% (5/70) to isoniazid (INH) and rifampicin. More ethambutol (EMB) and combined INH+EMB resistance was detected using MAS-PCR. CONCLUSION: MDR-TB is a problem in Tripura, and culture and phenotypic DST are required for diagnosis. MAS-PCR may provide an alternative rapid screening tool.

Malsoor virus, a novel bat phlebovirus, is closely related to severe fever with thrombocytopenia syndrome virus and heartland virus.

During a survey in the year 2010, a novel phlebovirus was isolated from the Rousettus leschenaultii species of bats in western India. The virus was identified by electron microscopy from infected Vero E6 cells. Phylogenic analysis of the complete genome showed its close relation to severe fever with thrombocytopenia syndrome (SFTS) and Heartland viruses, which makes it imperative to further study its natural ecology and potential as a novel emerging zoonotic virus.

Acute and chronic toxicity of copper on aquatic insect Chironomus ramosus from Assam, India.

Acute toxicity of copper (Cu) on Chironomus ramosus was determined by exposing third-instar larvae to graded concentrations of copper sulphate (CuSO4 x 5H2O). Median lethal concentrations (LC50) of Cu as CuSO4 at 24, 48, 72 and 96 hr were determined as 3280, 1073.33,780 and 183 microg l(-1), respectively. For determining the effects of chronic toxicity, small first-instar larvae were individually exposed to sublethal concentrations of copper sulphate (1.0-18.0 microg l(-1)) for a period of 21 days. Discoloration and thinning of body were detected at 1 microg l(-1) and ventilation movements, pupation and adult emergence were significantly affected at 1.8 microg l(-1). At 10 microg l(-1) CuSO4 concentration, growth and tube-building activities of the larva were significantly different from the control.

Acute toxicity of endosulfan and malathion on Chironomus ramosus (Insecta: Diptera: Chironomidae) from north Cachar hills, Assam, India.

Acute toxicity tests for the pesticides endosulfan and malathion on the larvae of Chironomus ramosus were conducted. Median Lethal Concentration (LC50) values of endosulfan were 0.55 x 10(-2), 0.16 x 10(-2), 0.089 x 10(-2) and 0.036 x 10(-2) ppb respectively, while those for malathion were 0.139 x 10(-2), 0.054 x 10(-2), 0.019 x 10(-2) and 0.0032 x 10(-2) ppb respectively, at 24, 48, 72 and 96 hr. Thus Chironomus ramosus larvae were more sensitive to malathion at all hours of toxicity tests than endosulfan.

Role of virulence plasmid of Aeromonas hydrophila in the pathogenesis of ulcerative disease syndrome in Clarias batrachus.

Pathogenic Aeromonas hydrophila (strain VB21), a multiple-drug resistance strain contains a plasmid of about 21 kb. After curing of plasmid, the isolates became sensitive to antimicrobials, to which they were earlier resistant. The cured bacteria exhibited significant alterations in their surface structure, growth profile and virulence properties, and failed to cause ulcerative disease syndrome (UDS) when injected into the Indian catfish Clarias batrachus. Routine biochemical studies revealed that the plasmid curing did not alter the biochemical properties of the bacteria. After transformation of the plasmid into cured A. hydrophila the bacterium regained its virulence properties and induced all the characteristic symptoms of UDS when injected into fish. Thus, the plasmid plays a pivotal role in the phenotype, growth and virulence of A. hydrophila and pathogenesis of aeromonad UDS.

Quantitative determination of CGS 26214, a cholesterol lowering agent, in human plasma using negative electrospray ionization liquid chromatography-tandem mass spectrometry.

CGS 26214 is a synthetic cholesterol-lowering agent shown to be active in the rat, dog and monkey. The present work was conducted to develop a sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for quantitative determination of the compound in human plasma following clinical doses of 10-100 microg per day. A number of analytical challenges were encountered during the development of the assay. The compound was an ester and susceptible to hydrolysis under experimental conditions. A lower limit of quantitation of 50 pg/ml was needed due to the low clinical doses. Positive electrospray ionization of CGS 26214 yielded insufficient sensitivity needed for the studies. Consequently, LC-MS-MS conditions were optimized for the negative ion mode of detection. The sample preparation steps proved to be critical in order to reduce the possibility of microbore column (50 mm x 1.0 mm I.D.) obstruction, chromatographic deterioration, and matrix mediated electrospray ion suppression. The present method addressed the above issues. The method was accurate and reproducible and was successfully applied to generate plasma concentration-time profiles for human subjects after low oral doses of the compound.

Cryptococcosis in India: the awakening of a giant?

The menace of cryptococcosis has assumed global proportions over the years. The tropical climate of the Indian subcontinent offers a suitable environment for Cryptococcus neoformans, and the onslaught of the acquired immune deficiency syndrome (AIDS) pandemic since the early 1990s has substantially influenced the situation. Coupled with that are the advances in laboratory diagnostic techniques that have made accurate diagnosis increasingly available. These factors together have led to a sharp increase in the number of reported cases of cryptococcosis. This review attempts to present an overview of the status of cryptococcosis in India from its first description to the most recent times. The disease has been reported from almost all parts of the country. C. neoformans var. neoformans is predominantly found in clinical samples, while C. n. var. gattii infection has also been reported. An organ commonly involved is the central nervous system, among others. Both immunocompromised and apparently immunocompetent patients have been affected. Laboratory diagnosis is mostly by conventional methods, while effective therapeutic options are limited. Early diagnosis followed by institution of specific therapy, where possible, has effectively reduced mortality. Awareness of the disease and maintenance of a high index of clinical suspicion is required. An integrated approach to patient management with active interaction between the clinicians and the laboratory personnel would be highly beneficial. The wide variety of presentations of the disease seen in India suggests the possibility of occurrence of strain variation which needs to be investigated fully. Introduction of routine testing of antifungal susceptibility of clinical isolates is also important in order to obtain baseline data on susceptibility patterns and to predict in advance any shift in those patterns in the population. To maintain a high standard in all such endeavours, the establishment of an external quality control system is desirable.

Determination of the chiral isomers of CGS 26214, a synthetic thyromimetic agent, in human plasma using microbore chiral chromatography-tandem mass spectrometry.

CGS 26214 is a racemic compound having cholesterol-lowering activity in rats, dogs, and monkeys. This compound has two equipotent chiral components CGS 28934(-) and CGS 28935(+). An analytical challenge was to develop a sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the analysis of the chiral components in human plasma following clinical doses of 1 mg or less. Several issues had to be addressed in order to devise a LC/MS/MS assay for the above compounds. First, the compounds were esters and susceptible to hydrolysis under experimental conditions. Second, a lower limit of quantitation (LLOQ) of 0.4 ng/ml was needed. Third, positive electrospray ionization of CGS 26214 did not yield sufficient sensitivity needed for the studies in humans. Consequently, LC/MS/MS conditions were optimized for negative ion mode of detection. Fourth, sample preparation steps proved to be critical in order to reduce the possibility of microbore chiral-HPLC column (100 x 1.0 mm i.d.) obstruction, chromatographic deterioration, and matrix mediated electrospray ion suppression. Although the present method addressed the above challenges, its major drawback was limited sample throughput capability. Nonetheless, the method was successfully applied to generate plasma concentration-time profiles for human subjects after oral doses (0.9 mg) of the racemate as well as the optically pure isomers.

Determination of terbinafine (Lamisil) in human hair by microbore liquid chromatography/tandem mass spectrometry.

An analytical method for the determination of terbinafine (Lamisil(R)) in human hair was developed and validated. Human hair (10 mg) was hydrolyzed in 0.50 mL of 5.0 N sodium hydroxide for 1.5 h. The aqueous layer was extracted with 1.5 mL of n-hexane. The organic layer was separated and re-extracted with 0.20 mL of formic acid (12.5%)/2-propanol (85:15, v/v). The aqueous layer was separated and 0.010 mL of the aqueous extract was injected onto a reversed-phase microbore (50 x 1.0 mm i.d.) column for analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The instrument was equipped with an electrospray ionization (ESI) interface and operated in the positive ion mode of detection. Interday and intraday accuracy and precision were assessed from the relative recoveries of spiked samples analyzed on three different days. The method showed excellent specificity and ruggedness with a lower limit of quantitation of 10 ng/g (i.e., 10 ppb) using 10 mg of human hair.

Trace-level quantitation of iralukast in human plasma by microbore liquid chromatography/tandem mass spectrometry.

Iralukast (CGP 45715A) is a potent peptido-leukotriene antagonist that is active in various in vitro and animal models for the treatment of asthma. An analytical challenge was to develop a sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with a lower limit of quantitation (LLOQ) of 10 pg/mL for the analysis of iralukast when administered at low doses during clinical trials. Several issues had to be addressed in order to devise a LC/MS/MS assay for the above compound. First, iralukast appeared to be light sensitive and unstable at room temperature under acidic conditions. Second, a LLOQ of 10 pg/mL was needed to support several clinical trials. Third, positive electrospray ionization of iralukast did not yield the necessary sensitivity required for studies in humans. Consequently, LC/MS/MS conditions were optimized for the negative ion mode of detection. Fourth, sample preparation steps proved to be critical to reduce the possibility of microbore HPLC column (50 mm x 1.0 mm i.d.) obstruction, chromatographic deterioration, and matrix-mediated electrospray ion suppression. While our validated method addressed the above challenges, its major drawback was limited sample throughput capability. Nonetheless, plasma concentration-time profiles for patients with moderate asthma after oral administration of 200, 500, 1000, and 5000 microgram/kg/day of iralukast were successfully obtained.

Quantitative analysis of terbinafine (Lamisil) in human and minipig plasma by liquid chromatography tandem mass spectrometry.

A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the determination of terbinafine in human and minipig plasma has been developed and validated. The method used positive-ion mode for monitoring terbinafine, and used a stable isotope labelled terbinafine as the internal standard. Subsequent to acetonitrile protein precipitation, the supernatant was directly (unfiltered) injected onto the LC column (retention time approximately 4.3 min) for analysis. Interday and intraday accuracy and precision were assessed from the relative recoveries (observed concentration in percent of the nominal value) of spiked samples analyzed on three different days. The lower limit of quantitation (LLOQ) was 0.0679 ng/mL in human and minipig using a plasma sample volume of 0.08 mL. The method was fast, specific, and exhibited ruggedness. Furthermore, the use of turbulent flow chromatography (TurboFlow LC/MS/MS) coupled to mass spectrometry for direct analysis of terbinafine in plasma is discussed. The technique allowed direct introduction of plasma with satisfactory chromatographic peak shape and increased throughput.

Liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry enantiomeric separation of dl-threo-methylphenidate, (Ritalin) using a macrocyclic antibiotic as the chiral selector.

Vancomycin, a macrocyclic antibiotic, is an amphoteric glycopeptide produced by Streptomyces orientalis which has proven to be a viable chiral selector for high performance liquid chromatograph (HPLC) (D. W. Armstrong, Y. Tang, S. Chen, Y. Zhou, C. Bagwill and J-R. Chen, Anal. Chem. (1994; 66: 1473). While it is related to other glycopeptide antibiotics, vancomycin has a number of unique structural features, including 18 stereogenic centers, five aromatic rings, and two side chains one of which is a carbohydrate dimer. Therefore, a vancomycin-based stationary phase appears to be multimodal in that it can be utilized in both normal-phase and reversed-phase liquid chromatography. Consequently, the enantiomeric separation may be operative via several mechanisms, including pi-pi complexation, dipole stacking, inclusion, hydrogen bonding, or combinations of these interactions. LC/MS/MS is a powerful tool for quantitative analysis when evaluated on the basis of speed, specificity, reliability and sensitivity. For these reasons, the present paper explored the feasibility of bonded macrocyclic glycopeptide phases for chiral LC/MS/MS quantitative analysis. Methylphenidate was used as a model compound. A rapid chiral bioanalytical method (<7.5 min) for the determination of the enantiomers of methylphenidate was developed. A lower limit of quantification (LLOQ) of 87 pg/mL was attained for the human plasma assay. This is to our knowledge the first example of enantioselective reversed-phase LC/MS/MS for methylphenidate. The chiral column was relatively cost effective and exhibited excellent performance with no separation deterioration observed after approximately 2500 injections.

Disseminated candidosis in premature twins.

A unique case of disseminated candidosis in premature twins is presented where twin A developed disease soon after birth and died prior to the administration of antifungal therapy. On the other hand, twin B developed infection on the 26th day of birth but survived, though with sequelae (hydrocephalus), since he was promptly and accurately diagnosed and treated.

Structural characterization and sequence distributions of polysiloxanes using pyrolysis MS/MS.

Novel polysiloxanes, with 4-(dialkylamino)pyridine substituents, are characterized by pyrolysis tandem mass spectrometry. These polymers form abundant cyclic oligomeric ions under both desorption electron ionization (DEI) and desorption chemical ionization (DCI) conditions. Product MS/MS spectra of the cyclic ions reveal characteristic fragmentations under low energy collision activated dissociation. Protonated cyclic oligomers higher than the pentamer are mainly due to the proton bound dimers of lower oligomeric units. The cyclic oligomers are shown to have proton affinities greater than 1000 kJ/mole. It is proposed that thermal depolymerization occurs through an intramolecular siloxane bond rearrangement, which is in agreement with a previously proposed "loop mechanism". Markovian statistical calculations are applied to the DCI mass spectral data in order to determine the sequence distribution of siloxane copolymers. Application of this method show that the monomers in the copolymers examined are non-randomly distributed.

Structural characterization of poly(N-alkyl-4-vinylpyridinium) triflates using pyrolysis/tandem mass spectrometry.

Desorption chemical ionization (DCI) and desorption electron ionization (DEI) of homo- and co-polymers of N-alkyl-4-vinylpyridinium triflates having ethyl, n-hexyl and n-dodecyl groups as N-alkyl substituents, produce mass spectra that display oligomeric ions. These positively charged ions are singly-charged and result from cleavage of the polymer into neutral oligomers and the loss of a single triflate anion per oligomer. Analogous negatively charged ions, in which each neutral oligomer carries an extra triflate anion, are observed in the desorption chemical ionization mass spectra. Each oligomer within the available mass range is represented in the mass spectra. The formation of cluster ions in which a single, multiply-charged cation is associated with a number of singly-charged anions, as observed for these ammonium polysalts, is unusual. Five major and three minor series of positively charged ions are observed in DCI and DEI methods of ionization. Ions in the different series correspond either to cleavage at different bonds between the constituent monomers or to hydrogen transfer in different directions. Unique and structurally diagnostic fragmentation processes are observed in tandem mass spectrometry (MS/MS) experiments performed using collision activated dissociation of mass-selected oligomeric ions.

Leishmania donovani: purification and partial characterization of a glycophosphosphingolipid antigen expressed on promastigote surface.

A ceramide-anchored glycophosphosphingolipid antigen was isolated from the lipid extract of Leishmania donovani promastigotes. The affinity-purified glycolipid antigen contained galactose, mannose, myo-inositol, phosphate, ceramide, and hexosamine but no sialic acid. The phosphate group was present internally at the core of the structure: inositol (1-O)-phosphorylceramide. The phosphate group became susceptible to alkaline phosphatase only after alkali-catalyzed hydrolysis of the glycolipid.