ITPA polymorphisms do not predict additional risk beyond TPMT and NUDT15 for thiopurine-induced cytopenia in inflammatory bowel disease.

INTRODUCTION AND AIM: Thiopurine-related leukopenia is associated with polymorphisms in the thiopurine methyltransferase (TPMT) and nucleoside diphosphate-linked moiety X type motif 15 (NUDT15) genes. However, those polymorphisms explain only a fraction of thiopurine-related leukopenia. Our aim was to study the role of an inosine triphosphate pyrophosphatase (ITPA) polymorphism in patients with inflammatory bowel disease (IBD) and thiopurine-related leukopenia that was unexplained by the TPMT and NUDT15 polymorphisms. MATERIAL AND METHODS: We enrolled consecutive IBD patients on thiopurines (azathioprine or 6-mercaptopurine) from January 2019-March 2020, at a tertiary care center in North India. The presence of the ITPA (C.94C > A) polymorphism was evaluated in all patients, along with its association with thiopurine-related leukopenia. RESULTS: Of the 33 patients (from a total of 119 patients) that developed leukopenia, 8 had the TPMT (n = 1) or NUDT15 (n = 7) polymorphism. Of the remaining 111 patients, their mean age was 36.36 ±â€¯13.54 years and 57 (51.3%) were males. Twenty-five (21.01%) had unexplained leukopenia. The ITPA polymorphism was detected in 4 (16%) patients in the unexplained leukopenia group and 24 (27.9%) patients in the non-leukopenia group (p = 0.228). The odds ratio for predicting leukopenia with the ITPA polymorphism was 0.4921 (95% CI 0.1520-1.5830, p = 0.234). CONCLUSION: The ITPA (C.94C > A) polymorphism was frequently detected in the study population but was not predictive for leukopenia in patients with IBD on thiopurine therapy.

Modeling of live-birth rates and cost-effectiveness of oocyte cryopreservation for cancer patients prior to high- and low-risk gonadotoxic chemotherapy.

STUDY QUESTION: What is the live-birth rate (LBR) and cost-effectiveness of fertility preservation with oocyte cryopreservation (FP-OC) compared to expectant management in cancer patients age 25-40 based on estimated gonadotoxicity of treatments 5 years after cancer diagnosis? SUMMARY ANSWER: Oocyte cryopreservation prior to cancer treatment is more costly, yet more effective (producing more live births), than not undergoing oocyte cryopreservation but it is most beneficial for patients undergoing high-risk chemotherapy (HRC). WHAT IS KNOWN ALREADY: The decision to undergo FP prior to treatment is multifactorial and can be costly and delay treatment. Not all treatments carry the same gonadotoxicity and patients may choose to undergo FP-OC based on the probability of premature ovarian insufficiency, predicted outcomes and cost. A comprehensive model that incorporates age at diagnosis and toxicity of treatment to help guide patients in the decision to undergo FP-OC does not yet exist. STUDY DESIGN, SIZE DURATION: This study used a Decision Analysis Model to estimate effectiveness and cost of FP for cancer patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Age-based estimates of LBR and cost per live birth were calculated for ages 25-40 years based on gonadotoxicity of treatment. A decision analysis model was constructed using Treeage Pro 2015 with case base probabilities derived from national registries, practice guidelines and medical records from a national network of infertility practices (IntegraMed). MAIN RESULTS AND THE ROLE OF CHANCE: Compared to no FP-OC, FP-OC improved LBRs for women of all ages undergoing either low-risk chemotherapy (LRC) or HRC; however, it was most cost effective for women undergoing LRC at older ages or HRC at younger ages. Although FP-OC results in higher LBRs, it was always more costly. Using donor oocyte IVF can be a successful alternative to autologous FP-OC. LIMITATIONS REASONS FOR CAUTION: Decision tree results reflect probabilities of certain events and are compiled from multiple reputable sources but are not directly derived from a recruited cohort of patients. Outcomes are based on United States estimates and should be interpreted in the broader context of individual patient diagnoses, treatment care plans and country of origin. WIDER IMPLICATIONS OF THE FINDINGS: The development of this analytic model will help guide practitioners in their counseling of women from age 25 to 40 years, who are considering FP-OC at the time of cancer diagnosis. It provides a realistic pathway from diagnosis to LB and accounts for the majority of costs and outcome possibilities. STUDY FUNDING/COMPETING INTEREST(s): This study was partially funded by a grant from National Institute of Health (NIH)/National Institute of Child Health and Human Development (NICHD) (R01 HD67683) to A.Z.S. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Heterogeneous vancomycin-intermediate among methicillin resistant Staphylococcus aureus.

BACKGROUND: Hetero-resistance vancomycin intermediate Staphylococcus aureus (hVISA) is phenotype, which on in-vitro susceptibility test is vancomycin susceptible (VSSA) but has a minority population of vancomycin intermediate (VISA). hVISA is responsible for vancomycin treatment failure. Population Analysis Profile- Area under Curve (PAP-AUC) is a test for detection of hVISA; however, this test is unsuitable for clinical microbiology laboratory. Tests, such as Brain Heart Infusion Agar with 6 µg/ml vancomycin (BHIA6V), E test and Macromethod E Test (MET) are available; however reported to have variable results. METHODS: 58 clinical isolates of Methicillin resistant S aureus (MRSA) having MIC of vancomycin more than 1 µg/ml by E test and agar dilution were analyzed by PAP-AUC, BHIA6V and MET. RESULT: The prevalence of hVISA was 6.9%. hVISA isolates were having vancomycin E test MIC >2 µg/ml. Sensitivity of BHIA6V, MET and E test with MIC >2 µg/ml were 0.75, 0.67 and 1.0 respectively; however, positive predictive values (PPV) were 0.43, 0.4 and 0.27 respectively with PAP-AUC. PAP-AUC ratio correlated with MIC by E test and MET. CONCLUSIONS: There is need for screening MRSA isolates showing in-vitro vancomycin susceptibility ≤2 µg/ml by agar dilution method for detection of hVISA. PAP-AUC test is unsuitable for routine laboratory testing. BHIA6V, MET and E test can be used for screening, however have low PPV.

In vitro vancomycin susceptibility amongst methicillin resistant Staphylococcus aureus.

BACKGROUND: Vancomycin is drug of choice for treatment of Methicillin Resistant Staphylococcus aureus (MRSA) infections. S. aureus with reduced vancomycin susceptibility (SA-RVS) is on rise. Current guidelines of detection of SA-RVS are based on MIC (Minimum Inhibitory Concentration) by broth or agar dilution methods. Vancomycin MIC by E test (Epsilometer Test) is an alternative. A study was undertaken to know the prevalence of SA-RVS and compare vancomycin MIC by agar dilution and E test. METHODS: A prospective study was undertaken at tertiary care hospital; 232 clinical MRSA isolates were included. Vancomycin MIC was undertaken by agar dilution method and E test. RESULTS: All isolates were sensitive to Linezolid. Two MRSA isolates had vancomycin MIC ≥4 µg/ml; vancomycin MIC50 and MIC90 of MRSA isolates was 0.5 and 0.2 µg/ml respectively by agar dilution method. There was agreement over 93.5% isolates in vancomycin susceptibility by agar dilution and E test. E test had sensitivity and positive predictive value of 1.0 (CI - 0.34-1.0) and 0.5 (CI - 0.17-0.83) respectively compare to agar dilution method. CONCLUSIONS: MRSA isolates continues to be susceptible to vancomycin and Linezolid. E test was found equally suitable in initial screening for vancomycin susceptibility. Due to geographic variation in prevalence, there is need of ongoing surveillance of SA-RVC.

Emergence of VIM-2 metallo-beta-lactamase producing Ralstonia pickettii clinical isolate in India.

A multidrug-resistant clinical isolate of Ralstonia pickettii from a woman was analysed. Modified Hodge test was positive for carbapenemase production. Conjugation experiment revealed the presence of conjugative plasmid of >140 Kb size typed as IncN type. This is the first report of emergence blaVIM-2 in R. pickettii in India.

Allogeneic haematopoietic stem cell transplantation: the Army hospital experience.

BACKGROUND: We analysed the results of allogeneic haematopoietic stem cell transplantation (HSCT) in various genetic disorders, bone marrow failures and haematological malignancies done from 2002 to 2010 at the Army Hospital, Research and Referral, Delhi. METHODS: A total of 119 matched-related allogeneic- HSCTs (allo-HSCTs) were done in 114 patients (men 76, women 38) aged between 2 and 60 years. Peripheral blood stem cells (n=75) and bone marrow (n=43) were used as the source of stem cells. RESULTS: The overall survival was 62.3% (71/114) at a median follow-up of 34 months. Graft versus host disease (GVHD) was seen in 42 (36.8%) patients; grade III/IV acute GVHD in 17 (15%) and chronic GVHD in 24 (21%) patients. There were 4 (3.5%) graft rejections and one nonengraftment. The overall mortality was 37.7% (n=43) and the main causes of death were GVHD (32%), infections (26%), relapse (23%) and regimen-related toxicity (11%). CONCLUSION: Our results are comparable to published data in most disease conditions. With improvements in GVHD prophylaxis and better supportive care, we need to further reduce our mortality and morbidity.

External auditory canal paraganglioma: an atypical presentation.

OBJECTIVE: We present the first published description of a painful paraganglioma of the external auditory canal. Atypical histopathology made the diagnosis difficult. We discuss the potential pitfalls of clinical diagnosis and treatment of such a case. CLINICAL PRESENTATION: A 49-year-old woman presented with left-sided otalgia, hearing loss and tinnitus. Physical examination revealed a firm swelling arising from the superior portion of the left external auditory canal. A clinical diagnosis of otitis externa was made. INTERVENTION: There was minimal response to medical treatment. The swelling was aspirated, leading to brisk bleeding. A tumour was suspected from the computed tomography scan, and confirmed by a biopsy. The patient underwent excision of the paraganglioma. The histopathology was atypical, making diagnosis difficult. CONCLUSION: Such unusual masses of the external ear should always be borne in mind, especially when dealing with atypical presentations of commonly encountered diseases. Clinicians should have a low threshold for early intervention with imaging and biopsy.

Susac syndrome: outcome of unilateral cochlear implantation.

OBJECTIVE: Susac syndrome comprises a triad of vestibulocochlear dysfunction, retinopathy and multifocal encephalopathy, which is characterised pathophysiologically by microangiopathy of the ear, retina and brain. Diagnosis is confirmed by magnetic resonance imaging of the brain and ophthalmological examination, which reveals branch retinal artery occlusion. Hearing loss persists in 90 per cent of patients. We present a case of successful hearing rehabilitation by cochlear implantation in a young woman with this syndrome. CLINICAL PRESENTATION: A 36-year-old woman presented with neurological symptoms suggestive of encephalitis. She subsequently developed vestibulocochlear symptoms. The diagnosis was confirmed upon magnetic resonance imaging and fluorescein angiography, which showed multiple peripheral retinal arterial occlusions. Hearing loss was fluctuant but gradually progressive over nine months, to bilateral profound sensorineural hearing loss. INTERVENTION: A left cochlear implant was placed, with a good outcome. CONCLUSION: In this Susac syndrome patient, the outcome of cochlear implantation was encouraging, notwithstanding the possible involvement of retrocochlear pathways.

Allostery in Hsp70 chaperones is transduced by subdomain rotations.

Hsp70s (heat shock protein 70 kDa) are central to protein folding, refolding, and trafficking in organisms ranging from archaea to Homo sapiens under both normal and stressed cellular conditions. Hsp70s are comprised of a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). The nucleotide binding site in the NBD and the substrate binding site in the SBD are allosterically linked: ADP binding promotes substrate binding, while ATP binding promotes substrate release. Hsp70s have been linked to inhibition of apoptosis (i.e., cancer) and diseases associated with protein misfolding such as Alzheimer's, Parkinson's, and Huntington's. It has long been a goal to characterize the nature of allosteric coupling in these proteins. However, earlier studies of the isolated NBD could not show any difference in overall conformation between the ATP state and the ADP state. Hence the question: How is the state of the nucleotide communicated between NBD and SBD? Here we report a solution NMR study of the 44-kDa NBD of Hsp70 from Thermus thermophilus in the ADP and AMPPNP states. Using the solution NMR methods of residual dipolar coupling analysis, we determine that significant rotations occur for different subdomains of the NBD upon exchange of nucleotide. These rotations modulate access to the nucleotide binding cleft in the absence of a nucleotide exchange factor. Moreover, the rotations cause a change in the accessibility of a hydrophobic surface cleft remote from the nucleotide binding site, which previously has been identified as essential to allosteric communication between NBD and SBD. We propose that it is this change in the NBD surface cleft that constitutes the allosteric signal that can be recognized by the SBD.

A five year review of clinical profile in HSP.

To study the clinical profile of Henoch Schönlein Purpura [HSP] in children. A retrospective case series of 30 consecutive children with a diagnosis of HSP, with special focus on clinical manifestations. Two cases with unusual features are described in detail. Data of 19 boys and 11 girls with a mean age of 10.55 years was reviewed. Overall skin was involved in 100%, joints in 86.7%, GIT in 80% and renal system in 30% Two-thirds had palpable purpura at presentation. The mean duration of appearance of skin lesions after preceding joint and gastrointestinal symptoms was 8.6 days and 6.6 days respectively. Abdominal pain was the most common gastrointestinal symptom. Knee and ankle involvement occurred in more than 3/4th of the patients with arthritis. Vesciculobullous lesions were seen in two patients while one patient had rheumatic fever. Most children with HSP will have classical manifestation of the disease but diagnostic confusion can occur in those with atypical or absent cutaneous features at the onset.

Ecological niche of Cryptococcus neoformans var. grubii and Cryptococcus gattii in decaying wood of trunk hollows of living trees in Jabalpur City of Central India.

Cryptococcus neoformans var. grubii and C. gattii were repeatedly isolated from decaying wood of trunk hollows in living trees growing in Jabalpur City in Central India. The isolation of C. gattii has been reported from decayed wood inside trunk hollow of Tamarindus indica (15.6%), Mangifera indica (2.2%), Pithecolobium dulce (12.5%), Syzygium cumini (14%), and one from bark of S. cumini. C. n. var. grubii was isolated from decaying wood debris of T. indica (4.4%), M. indica (13.3%), Terminalia arjuna (25%), S. cumini (2%), Cassia fistula (4.5%), and two from bark of S. cumini. The two species [corrected] never co-occurred in the same hollow. C. gattii [corrected] isolates belonged to serotype B. [corrected] The data strongly supported the colonization of the pathogen in decaying wood hollow of all six-tree species. Evidence of this was found by repeated isolation up to 820 days. P. dulce is being reported for the first time as natural habitat of C. gattii and T. arjuna and C. fistula as natural habitat for C. n. var. grubii. M. indica is being reported for the second time as the natural habitat of both species [corrected] (C. n. var. grubii and C. gattii). The population density of these pathogens from decaying wood debris of various tree species ranged between 0.5 x 10(3) cells/g and 6 x 10(5) cells/g. The seasonal variation has been seen in isolation of these pathogens. [corrected] Our result further reinforce the recently emerging evidence that the natural habitat of C. n. var. grubii and C. gattii is more generalized.

Diffusion with attrition.

This article treats the problem of the sharp front observed when a diffusing substance interacts irreversibly with binding sites within the medium. The model consists of two simultaneous partial differential equations that are nonlinear and cannot be solved in closed form. The parameters are the diffusion coefficient D in the direction under consideration (x), the interaction constant k, the binding-site concentration mu and the boundary concentration of the diffusing ion c(0). Our aim is to develop methods to enable the estimation of these parameters from the experimental data. An analytical solution for the case k --> infinity, as found by others, is given first and then a finite element analysis package is used to obtain numerical solutions for the general case. Graphs are presented to illustrate the effects of the various parameters. Simple graphical procedures are described to compute mu and c (0). The position of the advancing front xi then provides, together with mu, a way to estimate D. A mathematical identity relating D and x and a second one involving D, k and t help to reduce the complexity of the problem. A new, measurable quantity S(t) is defined as [see text] where f is the total concentration (free + bound) of the diffusing ion at time t, and detailed plots are furnished that permit the computation of k directly from S(t), mu and D. The accuracy with which such methods can be expected to determine the various parameters of the model is considered at some length. Finally, in a concluding section, we simulate typical experimental data, examine the validity of our methods, and see how their accuracy is affected by controlled amounts of various kinds of noise.

Improvement of duty-cycle heating compensation in NMR spin relaxation experiments.

To reliably measure NMR relaxation properties of macromolecules is a prerequisite for precise experiments that identify subtle variations in relaxation rates, as required for the determination of rotational diffusion anisotropy, CSA tensor determination, advanced motional modeling or entropy difference estimations. An underlying problem with current NMR relaxation measurement protocols is maintaining constant sample temperature throughout the execution of the relaxation series especially when rapid data acquisition is required. Here, it is proposed to use a combination of a heating compensation and a proton saturation sequence at the beginning of the NMR relaxation pulse scheme. This simple extension allows reproducible, robust and rapid acquisition of NMR spin relaxation data sets. The method is verified with (15)N spin relaxation measurements for human ubiquitin.

A phase cycle scheme that significantly suppresses offset-dependent artifacts in the R2-CPMG 15N relaxation experiment.

R2-CPMG 15N relaxation experiments form the basis of NMR dynamics measurements, both for analysis of nano-pico second dynamics and milli-micro second dynamics (kinetics). It has been known for some time that in the practical limit of finite pulse widths, which becomes acute when using cryogenic probes, systematic errors in the apparent R2 relaxation behavior occur for spins far off-resonance from the RF carrier. Inaccurate measurement of R2 rates propagates into quantitative models such as model-free relaxation analysis, rotational diffusion tensor analysis, and relaxation dispersion. The root of the problem stems from evolution of the magnetization vectors out of the XY-plane, both during the pulses as well as between the pulses. These deviations vary as a function of pulse length, number of applied CPMG pulses, and CPMG inter-pulse delay. Herein, we analyze these effects in detail with experimentation, numerical simulations, and analytical equations. Our work suggests a surprisingly simple change in the phase progression of the CPMG pulses, which leads to a remarkable improvement in performance. First, the applicability range of the CPMG experiment is increased by a factor of two in spectral width; second, the dynamical/kinetic processes that can be assessed are significantly extended towards the slower time scale; finally, the robustness of the relaxation dispersion experiments is greatly improved.

Synchronous cultures from the baby machine. A model for animal cells.

A baby-machine system that produces newborn Escherichia coli cells from cultures immobilised on a membrane was developed many years ago in an attempt to attain optimal synchrony with minimal disturbance of steady-state growth, and a model designed to characterise the nature and quality of the synchrony of such cells in a quantitative manner has been published. The baby machine has now been adapted for animal cells, and the present article is an attempt to modify the model to include these cells as well. The model consists of five elements, giving rise to five adjustable parameters (and a proportionality constant): a major, essentially synchronous group of cells with ages distributed normally about zero; a minor, random component from a steady-state population on the membrane that had undergone only very little age selection during the elution process; a fixed background count, to allow for the signals recorded by the electronic particle counter produced by debris and electronic noise; a time-shift, to account for differences between time of cell division and end of sample collection; and the coefficient of variation of the interdivision-time distribution, taken to be reciprocal-normal. It is this last feature, a reciprocal-normal rather than a Pearson type III interdivision-time distribution, that distinguishes this version of the model from its predecessor. The model is fitted by unconstrained non-linear least-squares to data from three different leukemia cell lines. The standard errors of the parameters are quite small in all cases, making their estimates highly significant; the quality of the fit is striking. The five parameters of the model can be divided into two nuisance parameters, two that are associated with the methodology and one that describes an inherent property of the cell itself; it turns out that both methodology parameters are zero in all three data sets studied. We also discuss the partition of the transition-time dispersion between the age distribution of the newborn cells and the age distribution of dividing cells and show that a reliable estimate of the corresponding parameters requires an experiment that extends over at least two and a half doubling times.

Bacterial shape maintenance: an evaluation of various models.

In this article, we examine a large number of combinations of growth models, with separate attention to cell volume, cylindrical surface-area, polar caps, nascent poles, onset of constriction, precision of cell division and interdivision-time dispersion, for Escherichia coli cells growing in steady state at various doubling times. Our main conclusion is striking, and quite general: exponential cylindrical surface-area growth is not possible, irrespective of the behaviour of cell volume, the polar regions, the nascent poles, or any other feature of cell growth-such cells never reach steady state. The same is true of linear cylindrical surface-area growth, regardless of when during the cell cycle the doubling in growth rate takes place. Only after the introduction of feedback into the surface-area growth law, do the cultures attain steady state, all of them. The other components of the models contribute only marginally to the properties of the steady state. Thus, whether the feedback applies just to the cylindrical portion of the cell or to its entire surface area affects only the coefficient of variation of cell radius and the radius-volume correlation. The dynamics of old-pole maintenance, constant area or constant shape, influences the radius-length and radius-volume correlations and, to a much lesser extent, the coefficients of variation of cell radius and length; how the nascent poles grow, whether linearly or exponentially, does not seem to matter at all. The absolute dimensions of the cells are set by the growth rate of the culture and have almost no effect when the feedback is taken to apply to the entire cell surface area; when it is limited to the cylindrical portion of the cell, however, both radius-length and radius-volume correlations increase with increasing doubling time. Comparison with published values was inconclusive. The nature of cell surface-area growth has therefore been settled, but whether the volume increases by simple-exponential or by pseudo-exponential growth, or whether the old poles maintain a constant shape or a constant area during the cell cycle, can be determined only with more precise experimental data. The form of nascent-pole growth is not resolvable by present techniques.