REDD1 (regulated in development and DNA damage 1) modulates the glucocorticoid receptor function in keratinocytes.

Glucocorticoids (GCs) are widely used for the treatment of inflammatory skin diseases despite significant adverse effects including skin atrophy. Effects of GCs are mediated by the glucocorticoid receptor (GR), a well-known transcription factor. Previously, we discovered that one of the GR target genes, REDD1, is causatively involved in skin atrophy. Here, we investigated its role in GR function using HaCaT REDD1 knockout (KO) keratinocytes. We found large differences in transcriptome of REDD1 KO and control Cas9 cells in response to glucocorticoid fluocinolone acetonide (FA): both the scope and amplitude of response were significantly decreased in REDD1 KO. The status of REDD1 did not affect GR stability/degradation during self-desensitization, and major steps in GR activation-its nuclear import and phosphorylation at activating Ser211. However, the amount of GR phosphorylated at Ser226 that may play negative role in GR signalling, was increased in the nuclei of REDD1 KO cells. GR nuclear import and transcriptional activity also depend on the composition of GR chaperone complex: exchange of chaperone FKBP51 (FK506-binding protein 5) for FKBP52 (FK506-binding protein 4) being a necessary step in GR activation. We found the increased expression and abnormal nuclear translocation of FKBP51 in both untreated and FA-treated REDD1 KO cells. Overall, our results suggest the existence of a feed-forward loop in GR signalling mediated by its target gene REDD1, which has translational potential for the development of safer GR-targeted therapies.

Hyperacute Vasospasm After Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: Cerebral vasospasm after aneurysmal subarachnoid hemorrhage typically occurs 3-14 days after aneurysm rupture. We describe a series of patients who developed vasospasm within minutes of aneurysm rupture. This phenomenon, which we term, "hyperacute vasospasm," has been reported in animal models of SAH, but hitherto has been poorly described in humans. METHODS: Eleven patients were identified from an institutional registry who had aneurysmal rupture during catheter cerebral angiography between 1997 and 2009. We quantified the degree of vasoconstriction using vascular diameter index (VDI). The change in VDI (delta VDI or DVDI) was calculated by determining the difference in VDI before and after the procedure. We also examined the relationship between hyperacute vasospasm and delayed cerebral ischemia. RESULTS: Ten of eleven (91%) patients with intraoperative aneurysm rupture had cerebral vasoconstriction within minutes of intra-procedural aneurysmal rupture. Six of eleven patients (55%) with hyperacute vasospasm developed delayed cerebral infarction. CONCLUSIONS: Hyperacute vasospasm is likely common in patients with intraoperative aneurysm rupture and may be an unrecognized element of the natural history of aneurysmal subarachnoid hemorrhage. In this limited series, there was an association between hyperacute vasospasm and delayed cerebral infarction.

Asymptomatic reproductive tract infections/sexually transmitted infections among HIV positive women.

This study aims to highlight the importance of screening all HIV positive women for various reproductive tract infections/sexually transmitted infections (RTIs/STIs) irrespective of symptoms and to determine its occurrence in asymptomatic HIV positive women. Relevant specimens were collected for diagnosis of various RTIs/STIs. STIs were diagnosed in nearly one-third of the HIV positive asymptomatic patients which is quite high. The national strategy for STIs/RTIs control misses out large number of asymptomatic RTIs/STIs in HIV positive women which is responsible for silently transmitting these infections in the community. So this strategy should be modified to include screening of all HIV positives women irrespective of symptoms of STIs/RTIs.

Cryptococcus gattii infection in solid organ transplant recipients: description of Oregon outbreak cases.

Cryptococcus gattii was recognized as an emerging infection in the Pacific Northwest in 2004. Out of 62 total infections in Oregon since the outbreak, 11 were in solid organ transplant (SOT) recipients. SOT recipients were more likely to have disseminated disease and higher mortality than normal hosts, who mostly had isolated mass lesions. The median time from transplantation to C. gattii diagnosis was 17.8 months. The primary sites of infection were lung (n = 4), central nervous system (n = 3), or both (n = 4). The Oregon-endemic strain, VGII (subtypes IIa and IIc) was present in 10 of 11 patients; the median fluconazole minimum inhibitory concentration (MIC) was 12 µg/mL (range 2-32 µg/mL) for this strain. We found C. gattii infection among organ transplant recipients was disseminated at diagnosis, had low cerebrospinal fluid cryptococcal antigen titers, and was associated with an elevated fluconazole MIC and high attributable mortality.

Rising trend of antimicrobial resistance among Neisseria gonorrhoeae isolates and the emergence of N. gonorrhoeae isolate with decreased susceptibility to ceftriaxone.

CONTEXT: Gonorrhoea is one of the most common sexually transmitted infections (STI) in developing countries and is a global health problem. AIMS: To analyze the trend of antimicrobial susceptibility of Neisseria gonorrhoeae isolates over the years, in a tertiary care hospital of North India. SETTINGS AND DESIGN: The study population comprised males with urethritis and females with endocervicitis attending STI clinic of our hospital. MATERIALS AND METHODS: In our STI laboratory, all gonococcal isolates are subjected to antimicrobial susceptibility testing by disc diffusion method as per CLSI guidelines. ß-Lactamase production is determined by chromogenic cephalosporin test. Minimum Inhibitory Concentration (MIC) for ceftriaxone is determined by E-test. STATISTICAL ANALYSIS USED: Data were expressed as percentages. The differences in percentages were tested for statistical significance by using χ2 test and P values were determined. RESULTS: The percentage of penicillinase producing N. gonorrhoeae (PPNG) increased from 8% in 1995-96 to 20% in 2004-05 and 88% in 2011-2013. Quinolone-resistant N. gonorrhoeae (QRNG) showed a significant increase from 12% in 1995-96 to 98.3% in 2004-05, while 84% isolates were found to be QRNG by 2011-2013. In January 2013 we detected our first gonococcal isolate with decreased susceptibility to third-generation cephalosporins; Ceftriaxone, Cefixime and Cefpodoxime (MIC for ceftriaxone = 0.19 µg/ml). CONCLUSIONS: The results of our study highlighted an alarming increase in the percentage of PPNG and QRNG strains over the years. Emergence of N. gonorrhoeae isolates with decreased susceptibility to third-generation cephalosporins is a cause of concern and thus emphasises the importance of antimicrobial susceptibility testing.

Use of antibiotics in animal agriculture & emergence of methicillin-resistant Staphylococcus aureus (MRSA) clones: need to assess the impact on public health.

Widespread use of antibiotics in human, veterinary medicine and agricultural settings has played a significant role in the emergence of resistant MRSA clones due to selection pressure. MRSA has now become established in human population as well as in various animal species. An animal associated clone, MRSA ST 398 has been reported from animal foods and also from human infections in the community as well as from the health care associated infections. Clonal relationship between strains of animal and human origins are indicators of interspecies transmission of clones. Spread of these organisms may pose a great impact on public health if animal associated strains enter into the community and health care settings. Surveillance is important to correlate the genetic changes associated with their epidemiological shift and expansion to predict its impact on public health. Strict regulations on the use of antibiotics in humans as well as in animal food production are required to control the emergence of drug resistant clones. t0 his article reviews the information available on the role of antibiotics in emergence of MRSA strains, their epidemiological shift between humans and animals and its impact on the public health.

Comparison of two disc diffusion methods with minimum inhibitory concentration for antimicrobial susceptibility testing of Neisseria gonorrhoeae isolates.

BACKGROUND: A few studies are available comparing either minimum inhibitory concentration (MIC) values with the Clinical and Laboratory Standards Institute (CLSI) disc diffusion method or MIC with the Australian Gonococcal Surveillance Program (AGSP) method. AIM: This study was conducted with the aim to identify the most feasible and cost-effective method for antimicrobial susceptibility testing of Neisseria gonorrhoeae. MATERIALS AND METHODS: Antimicrobial susceptibility testing of N. gonorrhoeae was performed using, in parallel, the E-test for MIC determination and disc diffusion by CLSI and AGSP techniques, and were compared. Susceptibility to penicillin, ciprofloxacin, tetracycline, ceftriaxone and spectinomycin and cefixime were determined by CSLI and AGSP method and Kappa statistics used to analyse the data with SPSS software. RESULTS: All isolates were susceptible to ceftriaxone and spectinomycin by three methods. Ninety-nine (99%) strains were resistant to ciprofloxacin, while 1% showed intermediate susceptibility to ciprofloxacin by all methods. Statistically, there was a moderate level of agreement between the methods for penicillin. CONCLUSION: All three methods gave reproducible results. Although the media used in the disc diffusion by the AGSP method is easy and cheap to prepare, the CLSI method of disc diffusion testing is recommended for susceptibility testing of gonococcal isolates because of its feasibility and 100% accuracy, with MIC by E-test as the reference method.

Differential biochemical response of rat kidney towards low and high doses of NiCl2 as revealed by NMR spectroscopy.

Heavy metals are known for their associated nephrotoxicity and nickel is no exception. An integrated metabonomic approach, based on high-resolution (1) H NMR spectroscopy, was applied to determine the acute biochemical effects of NiCl(2) on the renal tissues of rats. Kidney homogenates from rats treated with NiCl(2) at two dose levels (4 and 20 mg kg(-1) b.w., i.p.) and those from controls were analysed using (1) H NMR spectroscopy and also assessed for antioxidant parameters at days 1, 3 and 5 post-dose. The major metabolite changes corresponding to nickel exposure were related to amino acids, osmolytes and energy metabolites. Differential responses were observed in (1) H NMR spectra with exposure to low and high doses of NiCl(2). For high doses, (1) H NMR spectral analysis revealed alterations in renal tissues, along with damage to the cortical and papillary region and depletion of renal osmolytes such as betaine, trimethyl amine oxide, myo-inositol and taurine, which persisted until day 5 post-dose. The metabolite profile of (1) H NMR spectra obtained from animals treated with lower dose of NiCl(2) initially increased as an immediate stress response and then showed signs of recovery with the passage of time. NMR spectral analysis was well corroborated with histopathological and oxidative stress results. Nickel-induced oxidative stress was observed in both groups of animals with increased levels of antioxidant parameters at initial time points, but continued to increase in the high-dose group. The present study shows a huge potential of metabonomics for mapping organ-based metabolic response during heavy metal toxicity.

Typing of Methicillin resistant Staphylococcus aureus: a technical review.

Increasing prevalence of Methicillin-resistant Staphylococcus aureus (MRSA) worldwide is a growing public health concern. MRSA typing is an essential component of an effective surveillance system to describe epidemiological trends and infection control strategies. Current challenges for MRSA typing are focused on selecting the most appropriate technique in terms of efficiency, reliability, ease of performance and cost involved. This review summarises the available information on application, potential and problems of various typing techniques in discriminating the strains and understanding the epidemiology of MRSA strains. The phenotypic methods in general are easier to perform, easier to interpret, cost effective and are widely available, however less discriminatory. The genotypic methods are expensive and technically demanding, however more discriminatory. Newer technologies involving sequencing of various genes are coming up as broadly applicable and high throughput typing systems. Still there is no consensus regarding the single best method for typing of MRSA strains. Phage typing is recommended as first line approach in epidemiological investigation of MRSA strains. PFGE remains the gold standard for characterisation of outbreak strains. DNA sequencing methods including MLST, spa typing, SCCmec typing and toxin gene profile typing are more practical methods for detecting evolutionary changes and transmission events. The choice of typing technique further depends on the purpose of the study, the facilities available and the utility of data generated to answer a desirable research question. A need for harmonisation of typing techniques by following standard protocols is emphasised to establish surveillance networks and facilitate global MRSA control.

Lower genital tract infections in HIV-seropositive women in India.

OBJECTIVES: The presence of STD facilitates shedding of HIV and increases HIV-1 disease progression, possibly by increasing plasma viremia. Our aim was to study the presence of various associated Sexually transmitted disease/Reproductory tract infections in HIV-seropositive women in India. MATERIALS AND METHODS: The study included 40 HIV-seropositive women attending the antiretroviral therapy (ART) clinic at Lok Nayak Hospital. An informed consent was taken from all subjects. All cases were subjected to detailed gynecological examination and two types of swabs, i.e., a vaginal swab and a cervical swab were taken for STD/RTIs evaluation. The vaginal swabs were used for preparation of wet mount and KOH mount for diagnosis of trichomoniasis and candidiasis; to make a vaginal smear for Gram staining to diagnose bacterial vaginosis (BV) as per Nugent's criteria; for culture of aerobic bacteria and Candida spp. The cervical swab was used for isolation of Neisseria gonorrhoeae by culture and for detection of Chlamydia trachomatis antigen by Chlamydia microplate enzyme immunoassay kit (BIORAD). All data were analyzed using appropriate statistical tests. RESULTS: All 40 cases were evaluated for the presence of STD/RTIs associated with HIV infection. The women belonged to the reproductive age group (15-45 years) and majority (40%) of them were para 2. Most of the women (14, 35%) were in World Health Organization (WHO) stage I and maximum number (28, 70%) had their CD4 cell count more than 200 cells/ml. There was no significant correlation between WHO stage of HIV-seropositive women and their CD4 cell count (P=0.092). Out of 40 cases, 15 (37.5%) were on ART with maximum cases (53.3%) in WHO stage III. The duration of ART was more than 6 months in 9 (60%) cases. The most common presenting complaint was vaginal discharge in women with WHO stage II and III and 27.5% women showed vaginitis on per speculum examination. Laboratory tests showed high prevalence of BV (30%), mixed infection (30%), and candidiasis (10%) among HIV-seropositive women (P<0.001 in both). Women with BV were mostly in WHO stage I (38.4%) and stage II (36.3%), while those with mixed infection were mainly in WHO stage III (36.3%) and stage IV (40%).Women with candidiasis were mainly in WHO stage III. C. trachomatis antigen was found only in one subject (prevalence 2.5%). Both WHO stage and CD4 cell count had no significant correlation with presence of BV (P=0.056 and 0.063, respectively) and candidiasis (P=0.492 and 0.530, respectively). Maximum number of patients on ART had mixed infection (53.3%), while most of the patients (36%) not on ART had BV. There was no significant association between duration of ART and the presence of vaginal infections. CONCLUSIONS: The prevalence of gynecological symptoms and RTIs in HIV-seropositive women is high enough to warrant routine gynecologic evaluation and RTI screening in these patients. However, larger studies and trials are needed to evaluate the effects of ART on these abnormalities as well as to choose the best screening tool in HIV-seropositive women.

Development of TaqMan real-time polymerase chain reaction for the detection of the newly emerging form of carbapenem resistance gene in clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii.

PURPOSE: The newly emerging form of the so-called New Delhi Metallo-beta-lactamases (NDM-1) has been reported recently from patients worldwide and broadly thought as a potential source for the major global health problem. Thus, it is important to study the epidemiology of the so-called NDM-1 harbouring bacteria to prevent its further spread and to place effective control measures. The present study describes the use of the real-time polymerase chain reaction (PCR) assay for the detection of the bla NDM-1 gene using TaqMan probes among clinical isolates. MATERIALS AND METHODS: Clinical isolates of Escherichia coli (11 strains), Klebsiella pneumoniae (17 strains) and Acinetobacter baumannii (six strains) that were resistant to either of the carbapenems (meropenem or imipenem) were included in the study. The presence of carbapenemases in such strains was confirmed using the modified Hodge test. A real-time PCR assay was optimized for the detection of NDM-1 using a cloned synthetic gene fragment followed by testing of the clinical isolates. The findings were further confirmed using PCR and gene sequencing. RESULTS: TaqMan probe assay displayed a good detection limit with analytical sensitivity of the assay up to 10 copies of bla NDM-1 gene per reaction. The isolates of E. coli and K. pneumoniae revealed narrow range crossing point values (Cp values) between (12-17) cycles (mean Cp value 14), indicating number of bla NDM-1 gene copies of 106-108. The wider range of Cp values (15-34) cycles with a higher mean Cp value (23.6) was observed in A. baumannii with number of bla NDM-1 gene copies of 103-108. CONCLUSIONS: The study demonstrates that real-time PCR assay based on TaqMan chemistry is a useful technique for the detection of bla NDM-1 harbouring clinical isolates of E. coli, K. pneumoniae and A. baumannii. The assay has great precision in measuring the number of bla NDM-1 gene copies per specimen of DNA.

Drug susceptibility of Mycobacterium tuberculosis in patients with AIDS at a tertiary care hospital in northern India.

The study was done to determine the anti-tuberculosis drug resistance patterns of Mycobacterium tuberculosis (MTB) in AIDS patients. Fifty antiretroviral drug naïve new AIDS patients with clinical evidence of pulmonary tuberculosis and no previous history of tuberculosis were recruited. Baseline CD4 counts and plasma viral loads (PVL) were measured by flow cytometry and RT-PCR, respectively. Sputum samples were obtained from each patient and subjected to Ziehl-Neelsen staining and cultured on Lowenstein-Jensen medium and using the BACTEC 460 system (B460). Antimicrobial susceptibilities were tested in all isolates using the B460 gystem. The occurrence of MTB was found to be more common with a PVL>4 log10 copies/ml (odds ratio: 4.6). Of 15 MTB isolates, 8 (53.3%) had single drug resistance, 4 (26.7%) had multidrug resistance (MDR) and 1 (6.7%) had resistance to three drugs (non-MDR). Two isolates (13.3%) were sensitive to all the four drugs. Resistance to first line anti-tuberculosis drugs was found to be higher among AIDS patients with MTB.

Yeast identification in routine clinical microbiology laboratory and its clinical relevance.

Rapid identification of yeast infections is helpful in prompt appropriate antifungal therapy. In the present study, the usefulness of chromogenic medium, slide culture technique and Vitek2 Compact (V2C) has been analysed. A total of 173 clinical isolates of yeast species were included in the study. An algorithm to identify such isolates in routine clinical microbiology laboratory was prepared and followed. Chromogenic medium was able to identify Candida albicans, C. tropicalis, C. krusei, C. parapsilosis and Trichosporon asahii. Chromogenic medium was also helpful in identifying "multi-species" yeast infections. The medium was unable to provide presumptive identification of C. pelliculosa, C. utilis, C. rugosa, C. glabrata and C. hemulonii. Vitek 2 compact (V2C) differentiated all pseudohypae non-producing yeast species. The algorithm followed was helpful in timely presumptive identification and final diagnosis of yeast infections, including multi-species yeast infections.

Detection of Mycoplasma pneumoniae in children with lower respiratory tract infections.

Mycoplasma pneumoniae is known to be a major cause of lower respiratory tract infections (LRTIs) in children. We studied 75 children who had been hospitalized for community-acquired LRTIs for the detection of M. pneumoniae by serological analysis and polymerase chain reaction (PCR) to amplify a 277-base pair region of 16S rDNA gene of M. pneumoniae applied to throat swab specimens. Serological and/or PCR positive results diagnosed M. pneumoniae infection in 23 (30.7%) patients.

Staphylococcus aureus phage types and their correlation to antibiotic resistance.

CONTEXT: Staphylococcus aureus is one of the most devastating human pathogen. The organism has a differential ability to spread and cause outbreak of infections. Characterization of these strains is important to control the spread of infection in the hospitals as well as in the community. AIM: To identify the currently existing phage groups of Staphylococcus aureus, their prevalence and resistance to antibiotics. MATERIALS AND METHODS: Study was undertaken on 252 Staphylococcus aureus strains isolated from clinical samples. Strains were phage typed and their resistance to antibiotics was determined following standard microbiological procedures. STATISTICAL ANALYSIS: Chi square test was used to compare the antibiotic susceptibility between methicillin resistant Staph. aureus (MRSA) and methicillin sensitive S. aureus (MSSA) strains. RESULTS: Prevalence of MRSA and MSSA strains was found to be 29.36% and 70.65% respectively. Of these 17.56% of MRSA and 40.44% of MSSA strains were community acquired. All the MSSA strains belonging to phage type 81 from the community were sensitive to all the antibiotics tested including clindamycin and were resistant to penicillin. Forty five percent strains of phage group III and 39% of non-typable MRSA strains from the hospital were resistant to multiple antibiotics. CONCLUSION: The study revealed that predominant phage group amongst MRSA strains was phage group III and amongst MSSA from the community was phage group NA (phage type 81). MSSA strains isolated from the community differed significantly from hospital strains in their phage type and antibiotic susceptibility. A good correlation was observed between community acquired strains of phage type 81 and sensitivity to gentamycin and clindamycin.

Chlamydia trachomatis causing neonatal conjunctivitis in a tertiary care center.

Chlamydia trachomatis is considered a major aetiological agent of conjunctivitis in newborns. The objective of the present study was to determine the aetiology of neonatal conjunctivitis and clinico-epidemiological correlates of chlamydial ophthalmia neonatorum. Fifty-eight newborns with signs and symptoms of conjunctivitis were studied. Conjunctival specimens were subjected to Gram staining, routine bacteriological culture, culture for Neisseria gonorrhoeae and direct fluorescent antibody (DFA) staining for diagnosis of C. trachomatis infection. C. trachomatis was detected in 18 (31%) neonates. Findings suggest that since C. trachomatis is the most common cause of neonatal conjunctivitis, routine screening and treatment of genital C. trachomatis infection in pregnant women and early diagnosis and treatment of neonatal Chlamydial conjunctivitis may be considered for its prevention and control.

Profile of direct walk-in and referred clients attending Integrated Counselling and Testing Centre.

The HIV pandemic has had a profound impact on the health and economic conditions of individuals, and people living with HIV/AIDS are faced with the task of maintaining optimal health status despite an increasing insult to their immune status. The aim of the present study was to study the profile of direct walk-in and referred patients attending the Integrated Counselling and Testing Centre (ICTC) of a tertiary care hospital, which may provide important clues to understanding the epidemiology of the disease in a particular region. The study included all the attendees of the ICTC referred from the hospital or direct walk-in from January 2007 to December 2007. Three rapid HIV tests were used and the samples showing positive results in all the three tests were declared HIV positive. The results were analysed to correlate between HIV positivity, age, sex, route of transmission and direct walk-in/referred patients. A low proportion of ICTC attendees (27%) in our study were direct walk-ins. As regards HIV positivity, 312 (8.3%) out of 2440 males and 164 (4.3%) out of 1315 females were HIV positive. Among the referred HIV-positive patients 162 were males and 62 females, whereas among the direct walk-in HIV-positive patients, 150 were males and 102 females. Integrated counselling and testing is now seen as a key entry point for HIV prevention. In addition to scaling up ICTC services, it is also important to raise awareness by aggressive health education programmes and integration of ICTC into various community organizations.

Histopathology for the diagnosis of infectious diseases.

Histopathological examination of tissue biopsies for the identification of infectious organisms is a very important diagnostic tool. Conventional culture confirmation of tissue biopsies often fail to identify any pathogen as, first of all, invariably most of the tissue samples that are collected and sent for culture isolation are inappropriately collected in formalin, which prevents pathogen growth in culture media. Inadequate processing like grinding, etc. further hinders isolation. Presence of inhibitors like dead tissue debris, fibers, etc. also delays isolation. Microbiologists often lack expertise in identifying infectious pathogens directly from tissue biopsies by microscopic visualization. This review therefore acquaints microbiologists with the various methods available for detecting infectious agents by using histological stains. On histopathological examination of the tissue biopsy once, it is determined that a disease is likely to be due to an infection and has characterized the inflammatory response and hence associated microorganisms should be thoroughly looked for. Although some microorganisms or their cytopathic effects may be clearly visible on routine haematoxylin- and eosin-stained sections, additional histochemical stains are often needed for their complete characterization. Highly specific molecular techniques, such as immunohistochemistry, in situ hybridization and nucleic acid amplification, may be needed in certain instances to establish the diagnosis of infection. Through appropriate morphologic diagnoses and interlaboratory communication and collaboration, direct microscopic visualization of tissue samples can thus be very helpful in reaching a correct and rapid diagnosis.