Phenotypic and Molecular Characterization of Plasmid- Encoded Extended Spectrum Beta-Lactamases Produced by Escherichia coli and Klebsiella spp from Lahore, Pakistan

Purpose: To investigate the distribution of plasmid-encoded extended spectrum beta-lacatamases (ESBLs) in Lahore, Pakistan using different phenotypic and molecular methods.Methods: Escherichia coli and Klebsiella spp were obtained over a period of nineteen months (June 2007 to December 2008). Both were tested by the double disk synergy test, combined disk test and  Epsiometer-test (E-test) to evaluate their ability to detect ESBLs. The genotypes of ESBLs were analyzed by monoplex polymerase chain reaction (PCR), multiplex PCR, DNA sequencing and isoelectric focusing.Results: 662 E. coli and 153 Klebsiella spp were analyzed. Among these isolates, 39.3 % E. coli and 26.1 % Klebsiella spp were positive for extended spectrum beta-lactamases (ESBLs).71.9 % E. coli and 79.6 % Klebsiella spp showed minimum inhibitory concentration (MIC) in the range > 32/0.064 = 500 μl/mL for cetatzidime/cetatzidime + clavulanic acid, while 66.5 % E. coli and 69.1 % Klebsiella spp revealed MIC in the range of > 16/0.016 = 1000 μl/mL for cefotaxime/cefotaxime + clavulanic acid. Antibiotic susceptibility testing revealed that imipemem, meropenem and tazocine were the most effective in the management of such infections. The most frequent genotype of ESBL was OXA (19.2 %) for E. coli and SHV (92.5 %) for Klebsiella spp. The highest genotypic combination found was the combination of TEM/OXA (44.2 %) for E. coli.Conclusion: The resistance of E. coli and Klebsiella spp-producing ESBLs in Pakistan is a serious issue, and TEM, OXA and SHV type ESBL were the most common genotypes. Some isolates produced two or three genotypes at a time. Multiplex PCR of ESBL may help in early detection as well as phenotypic antibiotic therapy of these infections.Keywords: Beta-lactamases, Escherichia coli, Klebsiella spp, Antibiotic susceptibility,  Plasmidencoded, Structural genes, Imipemem, Meropenem, Tazocin

Core Gene Expression and Association of Genotypes with Viral Load in Hepatitis C Virus (HCV) - Infected Patients in Punjab, Pakistan

Purpose: To determine genotypic distribution, ribonucleic acid (RNA) RNA viral load and express core gene from Hepatitis C Virus (HCV) infected patients in Punjab, Pakistan.Methods: A total of 1690 HCV RNA positive patients were included in the study. HCV genotyping was tested by type-specific genotyping assay, viral load, by real time polymerase chain reaction (PCR) and HCV core protein was expressed in E. coli. Antigenicity of core protein was confirmed by enzyme-linked immunosorbant assay (ELISA).Results: Out of total 1690 serum samples, type-specific PCR fragments were observed in 1482 (87.69 %) of the samples. In both genders, genotype 3a (55.44 %) was most prevalent followed by 3b (15.03 %), 1a (6.98 %) and 1b (3.14 %). Regionally, genotype 3a occurred most frequently in Jaranwala (59.72 %). Patients infected with genotype 3 had pre-treatment viral load values of 52.56, 15.79 and 31.65 %, while patients infected by other genotypes showed viral load values of 13.43, 35.27 and 51.3 % for low, intermediate and high categories of viral load, respectively. ELISA showed that core protein possessed greater antigenicity.Conclusion: HCV genotype 3a is the most prevalent genotype in Punjab, although the distribution of HCV genotypes in eight cities of Punjab was not uniform. HCV core protein used to develop local screening assays may be more effective than current commercial assays.Keywords: Hepatitis C, Antigenicity, Genotyping, Viral load, Core gen

Diversity of Urinary Tract Pathogens and Drug Resistant Isolates of Escherichia coli in different age and gender Groups of Pakistanis

Purpose: This paper was mainly aimed to investigate drug resistance of the various urinary tract infection (UTI) pathogens from patients of different gender and age groups of Pakistanis. Method: For these purposes, urine samples of 109 patients were analyzed. Samples were screened on CLED agar. Antimicrobial susceptibility testing was performed by Kirby Bauer\'s disc diffusion method. Isolated colonies were processed for biochemical characterization and antibiotic sensitivity to ampicillin, amikacin, augmentin, ceftazidime, ceftriaxone, cefuroxime, cotrimoxazole, ciprofloxin, imipenem, meropenem, tazocine, trimethoprim, gentamicin and nitrofuratoin. Result: E.Coli was found to be the most frequent causative agent of UTIs (66%) followed by Enterococci (8.3%), Candida spp. and Pseodomonas spp. (7.3% each), Klebsiella spp. (5.5%) and Enterobacter spp. (2.7%). Proteus. and Morgenella species were found in less than 1% of the cases. E.coli showed variable antimicrobial resistance to different antibiotics as 92%, 86%, 80%, 62%, 47%, 20% and 4% of the isolates were found to be resistant to ampicillin, cotrimoxazole, ciprofloxin, gentamicin, nitrofuratoin and amikacin, respectively. Conclusion: The most effective in vitro agents were found to be amikacin followed by gentamicin (among the parenterals), and ciprofloxin among the orally administratered ones. A higher prevalence of UTIs was observed in the female population and E.coli showed no resistance to nitrofuratoin in age groups of 50+ and 70+ in both genders. Keywords: Urinary tract infections, Age, Gender, Resistant microbes, E.coli. Tropical Journal of Pharmaceutical Research Vol. 7 (3) 2008: pp. 1025-103

Effect of temperature anisotropy on various modes and instabilities for a magnetized non-relativistic bi-Maxwellian plasma

Using kinetic theory for homogeneous collisionless magnetized plasmas, we present an extended review of the plasma waves and instabilities and discuss the anisotropic response of generalized relativistic dielectric tensor and Onsager symmetry properties for arbitrary distribution functions. In general, we observe that for such plasmas only those electromagnetic modes whose magnetic field perturbations are perpendicular to the ambient magneticeld, i.e.,B1 \perp B0, are effected by the anisotropy. However, in oblique propagation all modes do show such anisotropic effects. Considering the non-relativistic bi-Maxwellian distribution and studying the relevant components of the general dielectric tensor under appropriate conditions, we derive the dispersion relations for various modes and instabilities. We show that only the electromagnetic R- and L- waves, those derived from them and the O-mode are affected by thermal anisotropies, since they satisfy the required condition B1\perpB0. By contrast, the perpendicularly propagating X-mode and the modes derived from it (the pure transverse X-mode and Bernstein mode) show no such effect. In general, we note that the thermal anisotropy modifies the parallel propagating modes via the parallel acoustic effect, while it modifies the perpendicular propagating modes via the Larmor-radius effect. In oblique propagation for kinetic Alfven waves, the thermal anisotropy affects the kinetic regime more than it affects the inertial regime. The generalized fast mode exhibits two distinct acoustic effects, one in the direction parallel to the ambient magnetic field and the other in the direction perpendicular to it. In the fast-mode instability, the magneto-sonic wave causes suppression of the firehose instability. We discuss all these propagation characteristics and present graphic illustrations

Etiology of Pediatric Meningitis in West Africa Using Molecular Methods in the Era of Conjugate Vaccines against Pneumococcus, Meningococcus, and Haemophilus influenzae Type b

Despite the implementation of effective conjugate vaccines against the three main bacterial pathogens that cause meningitis, Streptococcus pneumoniae, Haemophilus influenzae type b (Hib), and Neisseria meningitidis serogroup A, the burden of meningitis in West Africa remains high. The relative importance of other bacterial, viral, and parasitic pathogens in central nervous system infections is poorly characterized. Cerebrospinal fluid (CSF) specimens were collected from children younger than 5 years with suspected meningitis, presenting at pediatric teaching hospitals across West Africa in five countries including Senegal, Ghana, Togo, Nigeria, and Niger. Cerebrospinal fluid specimens were initially tested using bacteriologic culture and a triplex real-time polymerase chain reaction (PCR) assay for N. meningitidis, S. pneumoniae, and H. influenzae used in routine meningitis surveillance. A custom TaqMan Array Card (TAC) assay was later used to detect 35 pathogens including 15 bacteria, 17 viruses, one fungus, and two protozoans. Among 711 CSF specimens tested, the pathogen positivity rates were 2% and 20% by the triplex real-time PCR (three pathogens) and TAC (35 pathogens), respectively. TAC detected 10 bacterial pathogens, eight viral pathogens, and Plasmodium. Overall, Escherichia coli was the most prevalent (4.8%), followed by S. pneumoniae (3.5%) and Plasmodium (3.5%). Multiple pathogens were detected in 4.4% of the specimens. Children with human immunodeficiency virus (HIV) and Plasmodium detected in CSF had high mortality. Among 220 neonates, 17% had at least one pathogen detected, dominated by gram-negative bacteria. The meningitis TAC enhanced the detection of pathogens in children with meningitis and may be useful for case-based meningitis surveillance

Pediatric Bacterial Meningitis Surveillance in Nigeria From 2010 to 2016, Prior to and During the Phased Introduction of the 10-Valent Pneumococcal Conjugate Vaccine

Background: Historically, Nigeria has experienced large bacterial meningitis outbreaks with high mortality in children. Streptococcus pneumoniae (pneumococcus), Neisseria meningitidis (meningococcus), and Haemophilus influenzae are major causes of this invasive disease. In collaboration with the World Health Organization, we conducted longitudinal surveillance in sentinel hospitals within Nigeria to establish the burden of pediatric bacterial meningitis (PBM). Methods: From 2010 to 2016, cerebrospinal fluid was collected from children <5 years of age, admitted to 5 sentinel hospitals in 5 Nigerian states. Microbiological and latex agglutination techniques were performed to detect the presence of pneumococcus, meningococcus, and H. influenzae. Species-specific polymerase chain reaction and serotyping/grouping were conducted to determine specific causative agents of PBM. Results: A total of 5134 children with suspected meningitis were enrolled at the participating hospitals; of these 153 (2.9%) were confirmed PBM cases. The mortality rate for those infected was 15.0% (23/153). The dominant pathogen was pneumococcus (46.4%: 71/153) followed by meningococcus (34.6%: 53/153) and H. influenzae (19.0%: 29/153). Nearly half the pneumococcal meningitis cases successfully serotyped (46.4%: 13/28) were caused by serotypes that are included in the 10-valent pneumococcal conjugate vaccine. The most prevalent meningococcal and H. influenzae strains were serogroup W and serotype b, respectively. Conclusions: Vaccine-type bacterial meningitis continues to be common among children <5 years in Nigeria. Challenges with vaccine introduction and coverage may explain some of these finding. Continued surveillance is needed to determine the distribution of serotypes/groups of meningeal pathogens across Nigeria and help inform and sustain vaccination policies in the countr

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

PRKCA Polymorphism Changes the Neural Basis of Episodic Remembering in Healthy Individuals

Everyday functioning relies on episodic memory, the conscious retrieval of past experiences, but this crucial cognitive ability declines severely with aging and disease. Vulnerability to memory decline varies across individuals however, producing differences in the time course and severity of memory problems that complicate attempts at diagnosis and treatment. Here we identify a key source of variability, by examining gene dependent changes in the neural basis of episodic remembering in healthy adults, targeting seven polymorphisms previously linked to memory. Scalp recorded Event-Related Potentials (ERPs) were measured while participants remembered words, using an item recognition task that requires discrimination between studied and unstudied stimuli. Significant differences were found as a consequence of a Single Nucleotide Polymorphism (SNP) in just one of the tested genes, PRKCA (rs8074995). Participants with the common G/G variant exhibited left parietal old/new effects, which are typically seen in word recognition studies, reflecting recollection-based remembering. During the same stage of memory retrieval participants carrying a rarer A variant exhibited an atypical pattern of brain activity, a topographically dissociable frontally-distributed old/new effect, even though behavioural performance did not differ between groups. Results replicated in a second independent sample of participants. These findings demonstrate that the PRKCA genotype is important in determining how episodic memories are retrieved, opening a new route towards understanding individual differences in memory

Skin infection, housing and social circumstances in children living in remote Indigenous communities: testing conceptual and methodological approaches

BACKGROUND: Poor housing conditions in remote Indigenous communities in Australia are a major underlying factor in poor child health, including high rates of skin infections. The aim of this study is to test approaches to data collection, analysis and feedback for a follow-up study of the impact of housing conditions on child health. METHODS: Participation was negotiated in three communities with community councils and individual participants. Data were collected by survey of dwelling condition, interviews, and audit health centre records of children aged under seven years. Community feedback comprised immediate report of items requiring urgent repair followed by a summary descriptive report. Multivariate models were developed to calculate adjusted incidence rate ratios (IRR) for skin infections and their association with aspects of household infrastructure. RESULTS: There was a high level of participation in all communities. Health centre records were inadequate for audit in one community. The records of 138 children were available for development of multivariate analytic models. Rates of skin infection in dwellings that lacked functioning facilities for removing faeces or which had concrete floors may be up to twice as high as for other dwellings, and the latter association appears to be exacerbated by crowding. Younger children living in older dwellings may also be at approximately two-fold higher risk. A number of socioeconomic and socio-demographic variables also appear to be directly associated with high rates of skin infections. CONCLUSION: The methods used in the pilot study were generally feasible, and the analytic approach provides meaningful results. The study provides some evidence that new and modern housing is contributing to a reduction in skin infections in Aboriginal children in remote communities, particularly when this housing leads to a reduction in crowding and the effective removal of human waste