Introduction: Production of nitric oxide (NO) is one of the main responses elicited by a variety of immune cells such as macrophages (e.g. microglia, resident macrophages of brain), during inflammation. Evaluation of NO levels in the inflammatory milieu is considered important to the understanding of the intensity of an immune response; and has been performed using different methods including the Griess assay. To assay NO in culture, an appropriate number of cells are stimulated into an inflammatory phenotype. Common stimuli include lipopolysaccharide (LPS), IFN-γ and TNF-α. However, overt stimulation could cause cell cytotoxicity therefore an ideal concentration of LPS should be used. Objective: To set-up a model of BV-2 cell activation that allows the assay of detectable levels of NO. Optimization of BV-2 microglia cell density and LPS concentrations after stimulation by bacterial lipopolysaccharide (LPS) for the Griess assay is demonstrated in this study. Methods: BV-2 microglia were cultured at different cell densities, and treated with LPS at three concentrations (1, 5, 10 µg/ml). NO production in culture supernatants were then measured at 18, 24, 48 and 72 hours. Moreover, methyl tetrazolium assay (MTT) was also performed to ensure that NO measurement is performed at no-cytotoxic concentrations of LPS. Results and Conclusions: NO production follows a temporal pattern. The density of 25000 cells/ well was the ideal seeding density for NO evaluation in BV-2 cells. BV-2 stimulation by LPS is dose dependent, and NO levels are increased proportional to the LPS concentration up to 1.0µg/ml, whereas the higher LPS concentrations are associated with decreased cell viability may be caused by the high toxic levels of LPS or NO. Although Griess assay has been commonly used by the scientists, however, optimization of its parameters on BV-2 cells will be useful for the experiments which will be performed on this particular cell line. The optimized pattern of Griess assay on BV-2 cells was achieved in this study, hence easier and more practical for the future scientists to perform Griess assay on BV-2 cells.
Nitric Oxide

Introduction: Group B Streptococcus (GBS), infection and recurrence in newborns and pregnant women can lead to chronic medical illness resulting in significant morbidity, and mortality. Pathogenesis of GBS may be due to reasons such as activation of the immune system, followed by the production of inflammatory markers and toxic components by immune cells including macrophages. Methods: The studies on invasive and colonizing GBS strains inoculated either with peripheral or brain macrophages, the expression of nitric oxide (NO), cell viability, and CD40 were also measured by Griess assay, methyl tetrazolium assay (MTT), and flow cytometry, respectively. Furthermore, the clinical manifestations of the selected patients were also assessed for this study. Results: Outcome of inflammatory markers studies, after GBS inoculation indicated that, invasive GBS strains induced higher inflammatory markers in comparison to colonizing GBS strains. Furthermore, patients’ clinical data showed that patients with invasive GBS infections had severe condition unlike among patients with colonizing GBS strains. The fatality rate in patients with invasive GBS strain were 30.8% while there was no death among carriers. Conclusion: This study, aimed to understand the immune response to GBS, and strengthen the knowledge on GBS pathogenesis. It was concluded that invasive GBS strains not only showed higher expression of inflammatory markers on immune cells but also had higher pathogenesis effect in comparison to colonizing GBS strains.
Streptococcus agalactiae ; Pregnancy

Staphylococcus aureus is usually considered a colonizer but can result in infections under favourable conditions, especially in the healthcare setting. Healthcare workers can be colonized by S. aureus, and may transmit them to patients under their care. We conducted a cross sectional study to determine the prevalence of S. aureus nasal carriers among medical students in Universiti Putra Malaysia (UPM) (from January to June 2011). Our study involved 209 medical students comprising of 111 and 97 preclinical and clinical students respectively. A selfadministered questionnaire was distributed and nasal swabs were collected. Upon identification, the antibiotic susceptibility of the isolates was examined followed by categorical analysis (Chi-square and Fisher’s exact tests) with factors associated with S. aureus nasal carriage. Twenty one (10%) S. aureus strains were isolated from 209 nasal swab samples. 14 isolates were from pre-clinical students while the remaining seven were from clinical students. There was no significant association between gender, ethnicity, health status, skin infection and students’ exposure to hospital environment with S. aureus nasal carriage (p>0.05). Nineteen (90.5%) isolates were resistant to penicillin and there was also no significant association between penicillin resistant and the students’ groups. One (5.3%) isolate was resistant to erythromycin. There was no methicillin-resistant S. aureus isolated in this study.

SUMMARY Introduction: Patients suffering from diabetes mellitus (DM) frequently present with infected diabetic foot ulcers (DFU). This study was done to record the anatomical site and the grade of ulcers according to Wagner’s classification and to culture the microorganisms from the ulcers and determine their antibiotic sensitivity. Materials and methods: Prospective study was conducted on 77 diabetic patients who were admitted with DFU from June until December 2011. Patients with end stage renal failure, those who had previous vascular surgery on the involved limb, or hyperbaric oxygen or maggot therapy for the ulcers, or had unrelated skin diseases around the involved foot were excluded from the study. Specimens for culture were obtained by a sterile swab stick or tissue sample was taken from the wound with sterile surgical instruments. Results: Wagner’s grade III and IV ulcers were most common. Majority of the ulcers involved toes (48%). Gram negative microorganisms were predominantly isolated (71.1%). Gram positive microorganisms were less frequently cultured (27.7%). Fungus was cultured from one sample (1.2%). Gram negative microorganisms were sensitive to aminoglycosides, cephalosporins or β-lactamase inhibitors. More than 40% were resistant to ampicillin. Gram positive microorganisms were sensitive to cloxacillin. MRSA were sensitive to vancomycin. Conclusion: Empirical use of antibiotics should be curtailed to prevent development of drug resistant strains of microorganisms and MRSA. We suggest use of antiseptic solutions to clean the ulcers until antibiotic sensitivity report is available. Results of our altered treatment regimen we plan to publish in a later study.
Diabetic Foot ; Foot Ulcer

Introduction: The increasing trend of extensively drugresistant gram negative bacteria responsible for nosocomial infections has prompted resurgence colistin usage. Colistin-induced nephrotoxicity is a concern with disparity in the reported rates between previous studies. This study aims to evaluate colistin-induced nephrotoxicity among Malaysian population. Methods: The medical records of ICU patients receiving colistin therapy in Hospital Serdang and Hospital Sungai Buloh from 2010 to 2012 were retrospectively reviewed. Demographics data, treatment characteristic as well as culture result and creatinine level were documented. Nephrotoxicity was determined based on RIFLE criteria. Results: A total of 100 patients were included. Median daily dose, cumulative dose and duration of colistin therapy were 3.0 MIU (IQR: 4, range 1-12), 17.8 MIU (IQR: 31.5, range 2-180) and seven days (IQR: 4, range 1-30). Nephrotoxicity was found in 23% of the study population. All cases were reversible but marginally associated with higher mortality. No statistical association exist between age, gender and race as well as administration routes with nephrotoxicity by univariable analysis. The association of dose and duration with nephrotoxicity was also not significant by univariable analysis. After adjustment for confounders, statistical association between the independent variables and dependent variable remains not significant. Conclusion: Lower dose and shorter duration in local settings contribute to lack of association between colistin therapy and nephrotoxicity in this study. Higher dosing regimen with loading dose application has been introduced in the latest National Antibiotic Guideline. Further evaluation of colistin-induced nephrotoxicity and potential risk factors is therefore warranted.
Colistin ; Intensive Care Units

The aim of this study was to study the genotype of a hospital collection of Group B Streptococcus (GBS) from invasive and non-invasive sites. Fifty-one pre-characterised human of GBS were re-identified and further analysed by multilocus sequence typing (MLST) in relation to previously published serotypes. Fifteen sequence types (ST) were found with ST1 being the most predominant. ST1 was also associated with majority of the invasive isolates. The genotypic distribution patterns of GBS in this study were largely in agreement with previous reports from other countries indicating the tendency of certain genotypes to prevail in human infection settings.

Aims: To characterize the genotypic distribution of mec complex, bla complex, methicillin-resistance level (cefoxitinMIC) and β-lactamase activity in carriage methicillin-resistant Staphylococcus species for a potential correlation. Methodology and results: Biochemical test, 30 µg cefoxitin diffusion disc test, cefoxitin E-test, mec and bla complexes distributions, Pbp2a and β-lactamase assays were conducted to characterize phenotypic and genotypic of MRSA and MRCoNS in our collection. Phylogenetic tree was constructed using MEGA6 software to trace the diversity of blaZ gene of MRSA and MRCoNS. Sixteen MRSA and nineteen MRCoNS were identified by biochemical tests followed by 30 µg cefoxitin antibiotic disc susceptibility test and mecA gene screening. Twenty nine isolates carry complete mecA genes (2.1 kb), incomplete mec regulator (negative or truncated) and positive Pbp2a assay for both MRSA and MRCoNS. Only MRCoNS SC177 isolate with cefoxitin MIC of 32 µg/mL carries complete mec complex. Thirty-one of thirty-five isolates carry complete bla complex (blaZ, blaRI, blaI) with 10 MRSA produce strong β-lactamase and cefoxitin MIC of ≥12 µg/mL. Only 4 MRCoNS with cefoxitin MIC of ≤8 µg/mL produce strong β-lactamase. The diversity of blaZ gene was demonstrated by phylogenetic analysis and unusual amino acid mutation at position 145 for MRSA SA60 isolate may compromise its β-lactamase activity with low cefoxitin MIC level (2 µg/mL). Conclusions, significance and impact of the study: Isolates that carry complete complete mecA gene were largely consistent with the expression of Pbp2a. Nevertheless, there is no clear correlation of mec regulator genes in relation to cefoxitin-MIC in both methicillin resistant (MR) Isolates that carry Staphylococcus species. On the other hand, various expression level of β-lactamase may correlate with cefoxitin-MIC level in MRSA as compared to MRCoNS.

Objeetive:To update the status of Gardnerella vaginalis (G.vaginalis) as a causative agent of bacterial vaginosis (BV) in Malaysia and to define its epidemiology,metronidazole resistance and virulence properties.Methods:It is a single-centre (Gynaecology clinic at the Hospital Kuala Lumpur,Malaysia) prospective study with laboratory-based microbiological follow up and analyses.Vaginal swabs collected from the patients suspected for BV were subjected to clinical BV diagnosis,isolation and identification of G.vaginalis,metronidazole susceptibility testing,vaginolysin and sialidase gene PCR,Piot's biotyping and amplified ribosomal DNA restriction analysis genotyping.Results:Among the 207 patients suspected for BV,G.vaginalis was isolated from 47 subjects.G.vaginalis coexisted with Trichomonas vaginalis and Candida albicans in 26 samples.Three G.vaginalis isolates were resistant to metronidazole.Biotyping revealed 1 and 7 as the common types.Amplified ribosomal DNA restriction analysis genotype Ⅱ was found to be more common (n =22;46％) than Ⅰ (n =12;25.53％) and Ⅲ (n =13;27.6％).All genotype Ⅰ and Ⅲ isolates carried the sialidase gene,while 91.6％ and 84.6％ contained the vaginolysin gene.Genotype Ⅰ was significantly associated with postgynaecological surgical complications and abortions (P =0.002).Conclusions:The existence of pathogenic G.vaginalis clones in Malaysia including drug resistant strains should not be taken lightly and needs to be monitored as these may bring more complications especially among women of child bearing age and pregnant women.

Introduction: Challenge arises for medical undergraduates as the subject of medicine is intricate and extensive. Although students come from the same pool of excellent academic background, the medical undergraduates are still prone to failure in exams, resulting in them repeating the year of study or even having the thought of changing to other courses. In order to cope with the programme, students may adopt learning approaches that would help them to go through the programme.Therefore, it would be interesting to explore the learning approaches of medical students in Universiti Putra Malaysia. Methods: The purposes of this study were to determine the learning approach of medical undergraduates of Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (FMHS, UPM) together with its associated factors, which were socio-demographic characteristics and learning environment. This research was a cross-sectional study where the sample size calculated was 554. Self-administered questionnaires were given to the respondents chosen by simple random sampling. The socio-demographic characteristics were analysed using descriptive statistics such as frequency and percentage. Chi-square test was used to analyse the association between the variables. Results: The response rate was 83% (460 respondents agreed to participate). The majority of the respondents were females (73.7%), Malay (67%), and in their clinical years of study (58.7%). Overall, most students preferred deep approach (DA) of learning (49.6%), followed by strategic approach (29.1%) and surface apathetic approach (21.3%) of learning. There were statistically significant associations between learning approach and gender (p=0.005), as well as between learning approach and year of study (p=0.037). Conclusion:Our study showed an association between learning approaches and year of study and gender. DA of learning was the preferred learning approach in medical students at FMHS, UPM. This approach of learning, where students learn to understand the subject matter, may result in students become effective learners. Their understanding about the subject matter will be applicable to their clinical practice in the future.

Sporotrichosis is a relatively uncommon fungal infection in human as compared to other fungal infectiction such as tinea corporis or cruris. The fungus can be found in the environment and on plant matter. Sporotrichosis usually results from trauma to the skin followed by exposure to the fungus. It can also be spread by cat scratch or bite. Sporotrichosis usually presents as multiple skin nodules arranged in a linear fashion over the limbs, often with associated lymphadenopathy. This calls for importance of full physical examination and a high index of suspicion in order to diagnose this infection that can either involve the skin or the lymphatic channel or both. If misdiagnosed, treatment can be delayed and lead to prolonged suffering of the patient. This is an interesting case report about a 43-year-old woman who presented with left axillary swelling and decreased range of movement of the corresponding shoulder which was feared initially to be linked to breast cancer. However, good clinal acumen lead to the actual diagnosis of sporotrichosis.