Primary External Iliac Vein Leiomyosarcoma.

Leiomyosarcomas of vascular origin are uncommon. They arise from the smooth muscles of tunica media of major blood vessels. Most of the cases are present in women. We report one such case of external iliac vein leiomyosarcoma in a 77-year-old female and discuss the clinical, radiological, and histopathological findings and the available treatment options.

Structural and thermodynamic basis of amprenavir/darunavir and atazanavir resistance in HIV-1 protease with mutations at residue 50.

Drug resistance occurs through a series of subtle changes that maintain substrate recognition but no longer permit inhibitor binding. In HIV-1 protease, mutations at I50 are associated with such subtle changes that confer differential resistance to specific inhibitors. Residue I50 is located at the protease flap tips, closing the active site upon ligand binding. Under selective drug pressure, I50V/L substitutions emerge in patients, compromising drug susceptibility and leading to treatment failure. The I50V substitution is often associated with amprenavir (APV) and darunavir (DRV) resistance, while the I50L substitution is observed in patients failing atazanavir (ATV) therapy. To explain how APV, DRV, and ATV susceptibility are influenced by mutations at residue 50 in HIV-1 protease, structural and binding thermodynamics studies were carried out on I50V/L-substituted protease variants in the compensatory mutation A71V background. Reduced affinity to both I50V/A71V and I50L/A71V double mutants is largely due to decreased binding entropy, which is compensated for by enhanced enthalpy for ATV binding to I50V variants and APV binding to I50L variants, leading to hypersusceptibility in these two cases. Analysis of the crystal structures showed that the substitutions at residue 50 affect how APV, DRV, and ATV bind the protease with altered van der Waals interactions and that the selection of I50V versus I50L is greatly influenced by the chemical moieties at the P1 position for APV/DRV and the P2 position for ATV. Thus, the varied inhibitor susceptibilities of I50V/L protease variants are largely a direct consequence of the interdependent changes in protease inhibitor interactions.

Functional outcome of patients undergoing replantation of hand at wrist level-7 year experience.

BACKGROUND: Replantation is defined as reattachment of amputated limb using neurovascular and musculoskeletal structures in order to obtain recovery of limb. Re-vascularisation involves all the above steps in case of limb injuries that result in a near total amputation. AIM AND OBJECTIVE: To study the functional outcome of patients undergoing replantation of hand at wrist level. MATERIAL AND METHODS: This is a retrospective study of patients who underwent replantation of total amputation of hand at wrist level within a period of Jan 2003-June 2010. We evaluated post operative functional outcome compared to uninjured hand taking into consideration: 1. The patient's overall satisfaction with the hand. 2. Recovery of flexor and extensor function of thumb and fingers. 3. Recovery of thumb opposition. 4. Recovery of sensations in the median and ulnar nerve distribution. 5. Ability of surviving hand to perform daily tasks. RESULTS: There were total seventeen patients and age range was two years to 55 years. Out of 17 patients,16 were males. All the replantations were successful except for one. SUMMARY: The results showed that, although the replanted hands were never functionally as good as the contralateral hand the patients were able to perform most of the daily activities.

Hydrophobic core flexibility modulates enzyme activity in HIV-1 protease.

Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.

Mupirocin Resistance Among Methicillin Resistant Staphylococcus Isolates in a Tertiary Health Care Center.

INTRODUCTION: The evolution of antibiotics in the last century has revolutionized the field of medicine and led this field to higher level of success in treating mild to severe infections, but the inappropriate use of these life saving drugs has been accompanied with the appearance of resistant strains to these agents. AIMS & OBJECTIVES: The aim of the study was to determine the prevalence rate of high and low-level mupirocin resistance in methicillin resistant staphylococcus,and to find out resistance to other antibiotics. MATERIALS & METHODS: This study was conducted on 100 Staphylococcus isolates recovered from pus samples. Conventional disc diffusion tests were used for the detection of high and low level mupirocin resistance (mupirocin 5µg and 200µg discs) and for various other antimicrobials for example cephalexin, erythromycin, doxycycline, oxacillin, linezolid etc. Results: Outof 100 Staphylococcus isolates processed during the study period in the department of microbiology, 74 were Staphylococcus aureus (S.aureus) and 26 were Coagulase negative Staphylococcus (CoNS). Among S.aureus 43.4% were Methicillin resistant Staphylococcus aureus (MRSA) and 56.6% were Methicillin sensitive Staphylococcus aureus (MSSA), whereas among CoNS 42% were methicillin resistant and 58% were methicillin sensitive. We observed 6.75% of high level mupirocin resistance among Staphylococcus aureus and 19.23% among Coagulase negative staphylococcus. CONCLUSION: It was concluded that an inappropriate excessive use of mupirocin leads to a rapid increase in high-level resistance to mupirocin and other antibiotics in CoNS, affecting the treatment lines and success rate of infection control in hospital settings.

Role of Non Albicans Candida Spp. and Biofilm in Neonatal ICU.

BACKGROUND: Candida spp. remains the fungal species most commonly associated with biofilm formation. Increase in Candida infections in last decades has almost paralleled the increase and wide spread use of a broad range of medical implant devices mainly in population with impaired host defences. One of the most important characteristics of biofilms is their high level of resistance to antimicrobial drugs. AIMS AND OBJECTIVES: This study was conducted to know the prevalence of different Candida spp. causing blood stream infections and ability to form biofilm and to evaluate the co relation of biofilm with antifungal drug resistance. MATERIAL AND METHODS: The present study was conducted on 12464 blood samples for the identification and speciation of various Candida spp. causing blood stream infection over a period of one year. Antifungal susceptibility was performed as per clinical laboratory standard institute guidelines and biofilm formation was detected by method described by Christensen's et al. RESULTS: Out of total 12464 blood culture received, 1378 (11.05%) were culture positive rest and among culture positive 100 (7.25%) Candida isolates were recovered. C. tropicalis was the commonest (43%) species followed by C. albicans (41%), C. krusei (9%) and C. parapsilosis (7%). A total of 41 Candida isolates were biofilm producers and rest 59 isolates were non-biofilm producers. CONCLUSION: A changing trend of increased prevalence of non albicans Candida spp. was observed which were resistant to commonly used antifungal fluconazole. Multi drug resistance was more common in biofilm forming Candida isolates.

Case Report: Escherichia fergusonnii - Pathogen in Urinary Tract Infection.

Urinary tract infections are one of the leading cause of morbidity in admitted patients. Most commonly caused by Escherichia coli, but there are some variants which are commonly reported in urinary tract infection. This study was about to speciate such isolate like E.fergusonnii and find out its antibiogram.

Vancomycin and High Level Aminoglycoside Resistance in Enterococcus spp. in a Tertiary Health Care Centre: A Therapeutic Concern.

Aims. This study was aimed at knowing the prevalence of vancomycin and high level aminoglycoside resistance in enterococcal strains among clinical samples. Study Design. It was an investigational study. Place and Duration of Study. It was conducted on 100 Enterococcus isolates, in the Department of Microbiology, Pt. BDS PGIMS, Rohtak, over a period of six months from July to December 2014. Methodology. Clinical specimens including urine, pus, blood, semen, vaginal swab, and throat swab were processed and Enterococcus isolates were identified by standard protocols. Antibiotic sensitivity testing of enterococci was performed using Kirby-Bauer disc diffusion method. Results. High level gentamicin resistance (HLGR) was more common in urine samples (41.5%) followed by blood (36%) samples. High level streptomycin resistance (HLSR) was more common in pus samples (52.6%) followed by blood samples (36%). Resistance to vancomycin was maximum in blood isolates. Conclusion. Enterococci resistant to multiple antimicrobial agents have been recognized. Thus, it is crucial for laboratories to provide accurate antimicrobial resistance patterns for enterococci so that effective therapy and infection control measures can be initiated.

Fosfomycin use in multi drug resistant uropathogenic Escherichia coli.

OBJECTIVES AND AIMS: Escherchia coli isolated, from urine samples were studied for their antibiotic susceptibility patterns, with special reference to the new antimicrobial compound fosfomycin and their correlation with various virulence factors. MATERIAL AND METHODS: The mid stream urine samples received in the department were processed and identification was done by using the standard culture and identification techniques. The antibiotic susceptibility testing was done by modified Kirby-Bauer disk diffusion and the disk diffusion method was used to confirm the ESBL, AmpC, MBL production by the UPEC. Various virulence factors like hemolysin, haemagglutinaton, gelatinase, siderophore production, biofilm formation, serum resistance and hydrophobicity were detected. RESULTS: Fosfomycin was found to be most effective agent (100%) against uropathogenic E.coli followed by netilmicin (89.5%). The least effective agents were ampiciilin and cotrimoxazole. Twenty nine percent (29%) isolates were found to be multi drug resistant (MDR). CONCLUSIONS: The testing of the newer therapeutic agents like fosfomycin will add on to therapeutics for UTI's.

Changing trends of Candidemia and antifungal susceptibility pattern in a tertiary health care centre.

BACKGROUND: Bloodstream infections (BSI) caused by various Candida spp. are a significant cause of morbidity and mortality in hospitalized patients. An increasing proportion of device- related infections, particularly those involving the bloodstream and urinary tract, are being caused by Candida spp. AIMS AND OBJECTIVES: This study was conducted to evaluate the different species of Candida causing blood stream infections and their antifungal susceptibility. MATERIAL AND METHODS: The present study was conducted on 12464 blood samples received for culture, the samples were incubated at 37ºC for overnight and inoculated on culture plate next day. The growth on culture plates was identified by standard microbiological techniques and their antifungal susceptibility was put up as per Centre for Laboratory Standard Institute guidelines. RESULTS: Out of 12464 blood samples, isolation rate of Candida spp was 7.25%, which was higher in paediatric age group patients (89%) as compared to adults (11%). BSIs due to Candida spp. was significantly more common among ICUs (72%) than non-ICU settings (28%). C. tropicalis was the commonest (43%) species isolated followed by C. albicans (41%), C. krusei (9%) and C. parapsilosis (7%). All strains were 100% sensitive to Amphotericin B. CONCLUSION: There is a changing trend of increased isolation of non albicans Candida spp. than Candida albicans. It was common in ICUs settings and in paediatric age group. All isolates were found 100% sensitive to Amphotericin-B, C.krusei was 100% resistant to fluconazole followed by C. tropicalis 32.6% and C. parapsilosis 28.58%.

Biofilm and multidrug resistance in uropathogenic Escherichia coli.

CONTEXT: Escherichia coli is known as causative agent of urinary tract infections (UTIs) tends to form microcolonies in mucosa lining of urinary bladder known as biofilm. These biofilms make the organism to resist the host immune response, more virulent and lead to the evolution of antibacterial drug resistance by enclosing them in an extracellular biochemical matrix. AIMS: This study was done to know the association of various virulence factors and biofilm production in uropathogenic E. coli (UPEC) and antibiotic susceptibility pattern. SETTINGS AND DESIGN: This study was conducted in Pt. B.D. Sharma PGIMS, Rohtak, Haryana during a period of 1 year from January 2011 to December 2011. METHODS AND MATERIAL: Biofilm was detected by microtiter plate (MTP) method, and various virulence factors like hemolysin, hemagglutination, gelatinase, siderophore production, serum resistance, and hydrophobicity were detected. The antibiotic susceptibility testing was done by modified Kirby-Bauer disk diffusion and the disk diffusion method was used to confirm the ESBL, AmpC, MBL production by the UPEC statistical analysis used: The data were analyzed by using SPSS version 17.0. A two-sided P-value of less than or equal to 0·05 was considered to be significant. RESULTS: Biofilm production was found in 18 (13·5%) isolates, more commonly in females (two times). These isolates were found to be resistant to antibiotics common in use and were 100% MDR. CONCLUSIONS: Biofilm production makes the organism to be more resistant to antibiotics and virulent as compared to non-biofilm producers.

Acinetobacter lwoffii an emerging pathogen in neonatal ICU.

BACKGROUND: Acinetobacter species are ubiquitous in the environment and are important causative agent for nososcomial infection especially in immunocompromised patients. Multi drug resistant Acinetobacter lwoffii are emerging as a pathogen in neoanatal sepsis. AIMS AND OBJECTIVE: This study was aimed to evaluate the clinical and antibiotic profile of Acinetobacter lwoffii. MATERIAL AND METHODS: This study was done on blood samples from neonates admitted to neonatal intensive care unit during a period of one year from January to December 2012, who developed Acinetobacter infection. The diagnosis of isolates and antibiotic susceptibility testing was done by both conventional as well as by automated system. RESULTS: Out of total 13,133 blood samples received for culture, 1418(10.8%) were from NICU. Ninety (6.3%) isolates were found to be positive for the growth of Acinetobacter species. Of these isolates 31.11% were found to be Acinetobacter lwoffii, 68.9% were Acinetobacter baumannii calcaetius complex. Acinetobacter lwoffii isolates were most commonly sensitive to imepenem 16(57%), cotrimoxazole 9(32%), ciprofloxacin 6(21%) followed by amoxyclavulanic acid 2(7%) and cefuroxime 1(3.5%). CONCLUSION: Multi drug resistant Acinetobacter lwoffii infection is increasing particularly in premature and very low-birth weight neonates. Judicious and timely antibiotic use in NICUs are one of the important key in controlling multi-drug resistant Acinetobacter infection and improving clinical outcome.

Study of virulence factors of uropathogenic Escherichia coli and its antibiotic susceptibility pattern.

CONTEXT: Urinary tract infection (UTI) is one of the most common nosocomial infections, caused by Escherichia coli. This study determined the presence of virulence factors in the organism and correlates it with the multi-drug resistance (MDR). AIMS: The aim of the following study is to assess the virulence factors of uropathogenic E. coli and antibiotic susceptibility pattern. SETTINGS AND DESIGN: This was a prospective study conducted in the Department of Microbiology in PT. B. D. Sharma, PGIMS, Rohtak. SUBJECTS AND METHODS: The study was conducted over a period of 1 year. Urine samples received were processed as per standard microbiological procedures. Virulence factors such as hemolysin, hemagglutination, cell surface hydrophobicity, serum resistance, gelatinase and siderophore production were studied. The antimicrobial susceptibility was done as per Clinical and Laboratory Standard Institute Guidelines. STATISTICAL ANALYSIS USED: The data was analyzed by using SPSS(Statistical Package for the social sciences) IBM Corporation version 17.0. A two sided P ≤ 0.05 was considered to be significant. RESULTS: Hemolysin production was seen in 47.4%, hemagglutination in 74.8%, cell surface hydrophobicity in 61%, serum resistance in 59%, gelatinase in 67.5% and siderophore production in 88% isolates. Nitrofurantoin was found to be most effective followed by, gatifloxacin and gentamicin. Twenty nine percent (29.62%) isolates were MDR. CONCLUSIONS: Therefore, the knowledge of virulence factors of E. coli and their antibiotic susceptibility pattern will help in better understanding of the organism and in the treatment of UTI.

Comparison of ELISA and Microscopy for detection of Cryptosporidium in stool.

BACKGROUND: Cryptosporidiosis, a diarrheal disease caused by the protozoan parasite Cryptosporidium spp. has become recognized as one of the most common causes of water borne diseases in humans. AIMS AND OBJECTIVES: To compare the sensitivity of ELISA and Microscopy for detection of Cryptosporidium in stool samples Materials and Methods: The study was conducted in the Department of Microbiology of PT. B.D. Sharma PGIMS Rohtak, between January 2011 to june 2011 on 50 stool samples, which were processed for detection of cryptosporidial antigen by ELISA and detection of cysts by microscopy (Modified Ziehl and Nelsen staining). STUDY AND DESIGN: This was a prospective study conducted in the Department of Microbiology in PT. BD Sharma, PGIMS, Rohtak, India. RESULT: Out of total, 50 stool samples eighteen (36%) samples were found positive for Cryptosporidium cysts by microscopy in comparison to 3(6%) stool samples which were found positive for cryptosporidial antigen by ELISA. Samples found positive with ELISA were also positive with microscopy. Sensitivity, specificity, positive predictive value and negative predictive value for ELISA was 16.7%, 100%, 100% and 68% respectively. CONCLUSION: The study concludes that stool microscopic Modified acid fast staining is more sensitive method than ELISA for detection of Cryptosporidium in stool samples but the specificity of ELISA was more than microscopy.

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.

Molecular Basis for Drug Resistance in HIV-1 Protease.

HIV-1 protease is one of the major antiviral targets in the treatment of patients infected with HIV-1. The nine FDA approved HIV-1 protease inhibitors were developed with extensive use of structure-based drug design, thus the atomic details of how the inhibitors bind are well characterized. From this structural understanding the molecular basis for drug resistance in HIV-1 protease can be elucidated. Selected mutations in response to therapy and diversity between clades in HIV-1 protease have altered the shape of the active site, potentially altered the dynamics and even altered the sequence of the cleavage sites in the Gag polyprotein. All of these interdependent changes act in synergy to confer drug resistance while simultaneously maintaining the fitness of the virus. New strategies, such as incorporation of the substrate envelope constraint to design robust inhibitors that incorporate details of HIV-1 protease's function and decrease the probability of drug resistance, are necessary to continue to effectively target this key protein in HIV-1 life cycle.