Contributing role of TNF, IL-10, sTNFR1 and TNF gene polymorphisms in disease severity of leptospirosis.

The immune response is hypothesized as an important factor in the disease outcome of leptospirosis. Exaggerated immune response may promote tissue damage that lead to severe disease outcome. In this study TNF, IL-10, sTNFR1 levels were measured among sixty-two hospitalized leptospirosis confirmed patients in Sri Lanka. Thirty-one serum samples from healthy individuals were obtained as controls. PCR-RFLP method was used to identify TNF gene polymorphisms and to determine their association with TNF expression and disease severity in leptospirosis. TNF (p = 0.0022) and IL-10 (p < 0.0001) were found to be significantly elevated in leptospirosis patients, while sTNFR1 (p < 0.0001) was significantly suppressed. TNF was not significantly elevated in patients with complications while the anti-inflammatory cytokine IL-10 was significantly elevated among patients with complications (p = 0.0011) and with mortality (p = 0.0088). The ratio of IL-10 to TNF was higher among patients with complications (p = 0.0008) and in fatal cases (p = 0.0179). No association between TNF gene polymorphisms and TNF expression was detected due to the low frequency of heterozygous and mutated genes present in this study population. Thus the findings of the study show that elevated levels of IL-10 in the acute phase of disease could lead to severe outcomes and a high IL-10/TNF ratio is observed in patients with complications due to leptospirosis.

Essential Role of NADPH Oxidase-Dependent Production of Reactive Oxygen Species in Maintenance of Sustained B Cell Receptor Signaling and B Cell Proliferation.

Reactive oxygen species (ROS) are not only toxic substances inducing oxidative stress but also play a role as a second messenger in signal transduction through various receptors. Previously, B cell activation was shown to involve prolonged ROS production induced by ligation of BCR. However, the mechanisms for ROS production and ROS-mediated activation in B cells are still poorly understood. In this study, we demonstrate that BCR ligation induces biphasic ROS production in both mouse spleen B cells and the mouse B cell line BAL17; transient and modest ROS production is followed by sustained and robust ROS production at 2-6 h after BCR ligation. ROS production in the late phase but not in the early phase augments activation of signaling pathways, such as the NF-κB and PI3K pathways, and is essential for B cell proliferation. ROS production in the late phase appears to be mediated by NADPH oxidases (NOXes) because prolonged ROS production is inhibited by various NOX inhibitors, including the specific inhibitor VAS2870. BCR ligation-induced ROS production is also inhibited by CRISPR/Cas9-mediated deletion of either the Cyba gene encoding p22phox, the regulator of NOX1-4 required for their activation, or NOX3, whereas ROS production is not affected by double deficiency of the DUOXA1 and DUOXA2 genes essential for the activation of the NOX isoforms DUOX1 and DUOX2. These results indicate that NOXes play a crucial role in sustained but not early BCR signaling and suggest an essential role of NOX-dependent sustained BCR signaling in B cell activation.

Salmonella enterica serovar Paratyphi A isolated from a hard-to-heal diabetic ulcer: a case report.

Chronically infected diabetic wounds have a polymicrobial aetiology. However, Salmonella Paratyphi A is a very rare cause of wound infection. A 76-year-old female patient with type II diabetes presented with a wound on the left leg of two months' duration. The wound was painful, erythematous and a thick, foul-smelling discharge was present. There was a history of delayed wound healing. Salmonella Paratyphi A and Pseudomonas aeruginosa were isolated from the wound tissue. The patient was treated with cefuroxime and cloxacillin empirically and following the antibiotic susceptibility testing (ABST) report, ciprofloxacin was given for 10 days. The wound was treated with multiple debridements and topical antiseptic. On follow-up, the patient remained afebrile with subsiding discharge from the ulcer. This is the first reported case of Salmonella Paratyphi A from an infected diabetic ulcer in Sri Lanka and it serves to further define the spectrum of illnesses caused by this uncommon pathogen.

Type 2 diabetes mellitus and oral Candida colonization: Analysis of risk factors in a Sri Lankan cohort.

Aims: Oral candidiasis is a major oral manifestation of uncontrolled diabetes mellitus, and a number of cofactors are associated with the pathogenesis of this infection. Here, we describe the prevalence of oral Candida in a Sri Lankan cohort of type 2 diabetes mellitus and risk factors that predispose them to this common fungal infection. Methods: A case-control study was conducted in 250 diabetics with type 2 diabetes and 81 nondiabetic controls. Clinical and demographic data were collected using an interviewer administered questionnaire, and patient records. Oral rinse samples were collected to determine the candidal carriage, and the resultant yeast growth was quantified and speciated using multiplex-PCR and phenotypic analyses. Chi-square test (χ2 test) and Fisher exact test were used for the determination of the significant relationships between risk factors and oral candidiasis. Results: The oral prevalence of Candida species among both groups was similar (81%) although a significantly higher proportion of diabetics (32.8%) yielded >2000 CFU/mL of yeasts compared with only 12.3% of the healthy controls (p < .05). Significant associations were noted between oral candidal carriage amongst diabetics, and (i) denture wearing, (ii) female gender and (iii) cigarette smoking (all, p < .05). Amongst both groups, C.albicans was the most common Candida species isolated followed by C. parapsilosis, C. tropicalis and C. glabrata. Conclusions: The oral infestation of Candida in our Sri Lankan cohort of diabetics is significantly higher than their healthy counterparts, and co-carriage of multiple yeast species is a common finding in the study population. As there are no previous such reports of the latter phenomenon particularly from the Asian region it is noteworthy, mainly in view of the recent data on the emergence of drug-resistant yeast species the world over.

Association of tumor necrosis factor alpha gene polymorphisms with Helicobacter pylori infection in dyspeptic patients in Sri Lanka.

Single nucleotide polymorphisms present on the promoter sequence of the TNF-α gene may affect production of TNF-α, a pro-inflammatory cytokine, during immune responses. The presence of TNF-α polymorphisms is also reportedly associated with more severe manifestations of Helicobacter pylori infection. However, the frequency of TNF-α polymorphisms and the associated disease severity vary between different patient groups. In this study, gastric biopsies and blood specimens were collected from 138 patients with dyspepsia undergoing routine upper gastrointestinal endoscopy. Our institution's Ethics Review Committee approved the study and written informed consent was obtained from all participants. The presence of H. pylori was confirmed histologically in all patients. The frequency of TNF-α polymorphisms in the study cohort was investigated using PCR-restriction fragment length polymorphism and expression of serum TNF-α quantitated using a commercial ELISA assay. The proportions of selected TNF-α polymorphisms (TNF-α -238, -308 and -863) were similar in H. pylori-positive and -negative patients. Homozygous mutations of TNF-α polymorphisms were rarely detected in the study group. There was a significant difference in TNF-α concentrations between patients with mild chronic gastritis and TNF-α -308 GG genotype and patients with moderate to severe chronic gastritis (P = 0.008). It was not possible to identify an association between these genotypes and disease severity because of the low frequency of heterozygous and homozygous mutated genes in Sri Lankan patients with dyspepsia.

Do the Th17 Cells Play a Role in the Pathogenesis of Leptospirosis?

OBJECTIVES: The aim of this study was to determine the level of five different pro- and anti-inflammatory cytokines to study the inflammatory response of leptospirosis. MATERIALS AND METHODS: The serum cytokine levels of IL-10, IL-17A, IL-21, IL-23, and TNF-α were investigated in 57 patients with leptospirosis and 12 healthy controls using a commercially available ELISA kit (Mabtech, Sweden). Statistical analysis was done using Graphpad Prism. RESULTS: Elevation of serum IL-10 and IL-17A levels and significant elevation of serum IL-21 (p=0.002), IL-23 (p=0.002), and TNF-α (p=0.039) were observed among leptospirosis patients compared to the healthy control group. The two major complications observed among these patients were renal failure and liver involvement. Renal failure was significantly associated with elevation of IL-21 and IL-23, while patients with liver involvement had a significant elevation of IL-21, IL-23, and TNF-α. CONCLUSION: Elevation of IL-17A together with the significant elevation of IL-21 and IL-23 suggests a possible involvement of Th17 cells in the immunopathogenesis of leptospirosis.

Biosynthesized silver nanoparticles: are they effective antimicrobials?

BACKGROUND: Silver nanoparticles (AgNPs) are increasingly being used in medical applications. Therefore, cost effective and green methods for generating AgNPs are required. OBJECTIVES: This study aimed towards the biosynthesis, characterisation, and determination of antimicrobial activity of AgNPs produced using Pseudomonas aeruginosa ATCC 27853. METHODS: Culture conditions (AgNO3 concentration, pH, and incubation temperature and time) were optimized to achieve maximum AgNP production. The characterisation of AgNPs and their stability were evaluated by UV-visible spectrophotometry and scanning electron microscopy. FINDINGS: The characteristic UV-visible absorbance peak was observed in the 420-430 nm range. Most of the particles were spherical in shape within a size range of 33-300 nm. The biosynthesized AgNPs exhibited higher stability than that exhibited by chemically synthesized AgNPs in the presence of electrolytes. The biosynthesized AgNPs exhibited antimicrobial activity against Escherichia coli, P. aeruginosa, Salmonella typhimurium, Staphylococcus aureus, methicillin-resistant S. aureus, Acinetobacter baumannii, and Candida albicans. MAIN CONCLUSION: As compared to the tested Gram-negative bacteria, Gram-positive bacteria required higher contact time to achieve 100% reduction of colony forming units when treated with biosynthesized AgNPs produced using P. aeruginosa.

A sensitive and a rapid multiplex polymerase chain reaction for the identification of Candida species in concentrated oral rinse specimens in patients with diabetes.

OBJECTIVES: Oral candidiasis is being frequently recognized in patients with diabetes, and is associated with multiple pathogens including Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis. The aim of this study was to evaluate a usefulness of a Multiplex Polymerase Chain Reaction as a rapid diagnostic tool for identification of four oral Candida pathogens in patients with diabetes. MATERIALS AND METHODS: A multiplex PCR was optimized to identify four Candida species in concentrated oral rinse samples. Common reverse primer, ITS4 and four species-specific forward primers targeting ITS1 and ITS2 regions of yeast genome were used. Species-specific single amplicon were detected by agarose gel electrophoresis. Performance efficacy of multiplex PCR was compared with phenotypic identification. RESULTS: Out of 100 oral rinse samples, 72 were culture positive and of these 43 were at risk of oral Candida infection (>600cfu/ml). Multiple Candida species including C. albicans, C. parapsilosis and C. tropicalis were identified in 22 samples which had risk of oral Candida infection. In total, 85 patients were positive for Candida by multiplex PCR and of them 49 had multiple Candida species. All 43 colonized specimens were also positive by multiplex PCR. C. albicans was the most predominant organism (75/85) followed by C. parapsilosis (47/85), C. tropicalis (17/85) and C. glabrata (6/85). In specimens with multiple species, the two most common organisms were C. albicans and C. parapsilosis. Multiplex PCR yielded a sensitivity of 10 Candida cells/ml of oral rinse sample. CONCLUSIONS: Multiplex PCR is found to be rapid, sensitive and specific than phenotypic identification methods in discriminating multiple Candida species in oral rinse specimens.

The Effect of Nutritive and Non-Nutritive Sweeteners on the Growth, Adhesion, and Biofilm Formation of Candida albicans and Candida tropicalis.

OBJECTIVE: To determine the effect of glucose, sucrose, and saccharin on growth, adhesion, and biofilm formation of Candida albicans and Candida tropicalis. MATERIALS AND METHODS: The growth rates of mono-cultures of planktonic C. albicans and C. tropicalis and 1:1 mixed co-cultures were determined in yeast nitrogen broth supplemented with 5% (30 mM) and 10% (60 mM) glucose, sucrose, and saccharin, using optical density measurements at 2-h intervals over a 14-h period. Adhesion and biofilm growth were performed and the growth quantified using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The biofilm architecture was visualized using scanning electron microscopy. One- and two-way analysis of variance (ANOVA) was performed to analyse the differences among multiple means. RESULTS: The highest planktonic growth was noted in 5% glucose after 14 h (p < 0.05). No significant planktonic growth was observed in either concentration of saccharin. Both the concentrations of glucose and sucrose elicited significantly increased adhesion from MTT activity of 0.017 to >0.019 in mono- as well as co-cultures (p < 0.05), whilst the lower concentration of saccharin significantly dampened the adhesion. Maximal biofilm growth was observed in both species with the lower concentration of sucrose (5%), although a similar concentration of saccharin abrogated biofilm development: the highest MTT value (>0.35) was obtained for glucose and the lowest (>0.15) for saccharin. CONCLUSION: In this study, glucose and sucrose accelerated the growth, adhesion, and biofilm formation of Candida species. However, the non-nutritive sweetener saccharin appeared to dampen, and in some instances suppress, these virulent attributes of Candida.

Utility of modified Faine's criteria in diagnosis of leptospirosis.

BACKGROUND: Leptospirosis is a globally emerging zoonotic disease and an important public health threat in developing countries. Diagnosis of leptospirosis is mainly based on clinical presentations in resource poor countries. World Health Organization (WHO) has introduced "Faine's criteria" for diagnosis of leptospirosis. This study was conducted to evaluate the usefulness of modified Faine's criteria (with amendment) 2012 to detect leptospirosis in resource poor settings. METHODS: Blood samples of 168 patients who fulfilled the inclusion criteria admitted between January 2013 to January 2014 were tested by a commercial immunochromatographic assay (Leptocheck WB, India), microscopic agglutination test (MAT) and polymerase chain reaction (PCR) methods. Leptospirosis was confirmed by a single MAT titre ≥1:400 and / or by a positive PCR. Diagnosis of leptospirosis was made using the clinical, epidemiological and laboratory data according to modified Faine's criteria (with amendment) 2012. RESULTS: Leptospirosis was confirmed in 39 % (n = 66) by MAT and/or PCR. When modified Faine's criteria (MAT ≥ 1.400 &/ or PCR), was evaluated against LERG confirmed cases sensitivity, specificity, positive predictive value and negative predictive values were 95.45 %, 56.86 %, 58.88 %, 95.08 % respectively. The modified Faine's criteria with rapid immunochromatographic assay only had a sensitivity, specificity, positive predictive value and negative predictive value 89.39 %, 58.82 %, 58.42 %, and 89.55 % respectively. CONCLUSION: The modified Faine's criteria which utilized only immunochromatographic assay (leptocheck IgM) in Part C was found to be useful tool for diagnosing leptospirosis in a resource poor setting.

Culture media profoundly affect Candida albicans and Candida tropicalis growth, adhesion and biofilm development.

As there are sparse data on the impact of growth media on the phenomenon of biofilm development for Candida we evaluated the efficacy of three culture media on growth, adhesion and biofilm formation of two pathogenic yeasts, Candida albicans and Candida tropicalis. The planktonic phase yeast growth, either as monocultures or mixed cultures, in sabouraud dextrose broth (SDB), yeast nitrogen base (YNB), and RPMI 1640 was compared, and adhesion as well as biofilm formation were monitored using MTT and crystal violet (CV) assays and scanning electron microscopy. Planktonic cells of C. albicans, C. tropicalis and their 1:1 co-culture showed maximal growth in SDB. C. albicans/C. tropicalis adhesion was significantly facilitated in RPMI 1640 although the YNB elicited the maximum growth for C. tropicalis. Similarly, the biofilm growth was uniformly higher for both species in RPMI 1640, and C. tropicalis was the slower biofilm former in all three media. Scanning electron microscopy images tended to confirm the results of MTT and CV assay. Taken together, our data indicate that researchers should pay heed to the choice of laboratory culture media when comparing relative planktonic/biofilm growth of Candida. There is also a need for standardisation of biofilm development media so as to facilitate cross comparisons between laboratories.

Candida infection in oral leukoplakia: an unperceived public health problem.

OBJECTIVES: The study aimed to determine the proportion, known risk factors and etiology for Candida infection in leukoplakia lesions among patients with oral leukoplakia attending the Oral and Maxillofacial Clinic at a Tertiary Care Hospital in Sri Lanka. MATERIALS AND METHODS: Eighty clinically suspected oral leukoplakia patients were included. Two oral swabs each, from leukoplakia patients: one swab from the lesion and the other one from the contralateral unaffected corresponding area (as a control) were collected. Direct microscopy and culture followed by colony count and phenotypic identification were performed to identify pathogenic Candida species. RESULTS: Candida infection was seen in 47% of patients with oral leukoplakia. Candida albicans (94.7%) was the most common Candida species followed by Candida tropicalis (5.3%). Majority of Candida-infected lesions were seen in the buccal mucosa region. Alteration of taste (p = 0.021), having other oral lesions (p = 0.008), angular cheilitis (p = 0.024) and periodontitis (p = 0.041) showed a significant association with Candida-associated leukoplakia. Increasing age showed a significant tendency for Candida infection (p = 0.020). Smoking (p = 0.026) and betel-quid chewing (p = 0.006) were also found to be significantly associated, although alcohol consumption alone did not show a significant association. Oral leukoplakia patients who had all three habits: alcohol consumption, smoking and betel-quid chewing had a significant association with Candida infection (p = 0.004). CONCLUSIONS: Patients who had a combination of risk factors: smoking, betel-quid chewing and alcohol consumption were seen to have a significant association with Candida infection. Further betel-quid chewing alone and smoking singly was also significantly associated with Candida infection in oral leukoplakia.

Molecular characterisation and disease severity of leptospirosis in Sri Lanka.

Leptospirosis is a re-emerging zoonotic disease all over the world, important in tropical and subtropical areas. A majority of leptospirosis infected patients present as subclinical or mild disease while 5-10% may develop severe infection requiring hospitalisation and critical care. It is possible that several factors, such as the infecting serovar, level of leptospiraemia, host genetic factors and host immune response, may be important in predisposition towards severe disease. Different Leptospira strains circulate in different geographical regions contributing to variable disease severity. Therefore, it is important to investigate the circulating strains at geographical locations during each outbreak for epidemiological studies and to support the clinical management of the patients. In this study immunochromatography, microscopic agglutination test and polymerase chain reaction were used to diagnose leptospirosis. Further restriction fragment length polymorphism and DNA sequencing methods were used to identify the circulating strains in two selected geographical regions of Sri Lanka. Leptospira interrogans, Leptospira borgpetersenii and Leptospira kirschneri strains were identified to be circulating in western and southern provinces. L. interrogans was the predominant species circulating in western and southern provinces in 2013 and its presence was mainly associated with renal failure.

The Pattern of Substance Use and Characteristics of the Individuals Enrolled in Residential Treatment at Selected Rehabilitation Centers in Sri Lanka: A Descriptive Cross-Sectional Study.

Introduction: Substance use becomes censorious when it leads to harmful effects on individuals, their families, and the community. The nature of substance use in Sri Lankan context is poorly understood and empirical evidences are sparse. The study aimed to describe patterns of substance use and characteristics of the individuals enrolled in residential treatment at selected rehabilitation centers in Sri Lanka. Material and methods: A descriptive cross-sectional study was conducted among 205 individuals enrolled in selected rehabilitation centers. Pretested interviewer-administered questionnaire was used to collect data. Data were analyzed using descriptive statistics. Results: Most of the individuals who enrolled in residential treatment at selected rehabilitation centers were unmarried (n = 124, 60.5%), Sinhala (n = 186, 90.7%), Buddhist (n = 166, 81.0%), males (n = 202, 98.5%) and belonged to the young adult age (18-35 years) category (n = 178, 86.8%). All the participants were poly-drug users and cannabis was the most commonly used (n = 183, 89.3%) illicit drug followed by heroin (n = 172, 83.9%), methamphetamine (n = 150, 73.2%) and cocaine (n = 78, 38%). The most (n = 152, 74.1%) problematic substance for life was heroin. Most of the participants (n = 149, 72.7%) had used drugs several times per day. The mean duration of substance use was 7 ± 5 years. Participants (n = 177, 86.3%) reported that the substances were available in their residential areas and their friends (n = 197, 96.1%) were also using the substances. Conclusions: Pattern of substance use and characteristics of the individuals were unique in Sri Lanka and need to be considered when implementing and strengthening the programs for drug prevention and rehabilitation.

Importance of KIM-1 and MCP-1 in Determining the Leptospirosis-Associated AKI: A Sri Lankan Study.

BACKGROUND: Acute kidney injury (AKI) is one of most prevalent and serious complications of leptospirosis, a prevalent zoonotic disease in tropical countries. Prompt diagnosis of the leptospirosis-associated AKI is a challenge as there are no proper diagnostic tools that can identify patients in the early stage. Kidney injury molecule-1 (KIM-1) and monocyte chemoattractant protein-1 (MCP-1) are widely used novel AKI biomarkers that are studied in various disease conditions with AKI, but not in leptospirosis. Thus, this study is aimed at seeking the importance of KIM-1 and MCP-1 in determining the leptospirosis-associated AKI. METHODS: Leptospirosis-suspected patients who were admitted to medical wards of two selected hospitals in the Western province of Sri Lanka were recruited. Leptospirosis was confirmed by three diagnostic tests: PCR, MAT, and culture, and the status of AKI was determined by Kidney Disease Improving Global Outcomes (KDIGO) criteria. RESULTS: Of 170 leptospirosis-suspected patients, 79 were leptospirosis confirmed, and among them, 24.05% of patients were diagnosed to have AKI according to KDIGO criteria. Median serum KIM-1 (p < 0.0001), urine KIM-1 (0.0053), serum MCP-1 (0.0080), and urine MCP-1 (0.0019) levels in those developing AKI were significantly higher than in patients not developing AKI. The biomarker levels associated with leptospirosis AKI had AUC-ROC of 0.8565, 0.7292, 0.7024, and 0.7282 for serum KIM-1, urine KIM-1, serum MCP-1, and urine MCP-1, respectively. CONCLUSION: This study revealed serum KIM-1 as a promising marker for leptospirosis-associated AKI among the tested biomarkers. Thus, further validation is recommended with a larger study group.

Correction to: Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel­based DNA profiling method.

An amendment to this paper has been published and can be accessed via the original article.

Graphene Oxide-Based Nanocomposite for Sustained Release of Cephalexin.

A sustained-release carrier system for the drug cephalexin (CEF) using functionalized graphene oxide is reported. PEGylation of GO (GO-PEG) and successful loading of CEF into PEGylated graphene oxide (GO-PEG-CEF) nanoconjugate are confirmed by Fourier transform infrared spectroscopy, Raman spectroscopy, and thermogravimetric analysis. Encapsulation efficiency of 69% and a loading capacity of 19% are obtained with the optimized formulation of GO-PEG-CEF. In vitro CEF release profiles show an initial burst release followed by a more sustained release over a 96 h period with cumulative release of 80%. The half maximal inhibitory concentration (IC50) values have both dose- and time-dependent antibacterial activity for GO-PEG-CEF against both gram-positive and gram-negative bacteria while pure CEF showed only dose-dependent antibacterial activity. The minimum inhibitory concentration values of GO-PEG-CEF are 7.8 and 3.9 µg/mL against S. aureus and B. cereus, respectively, while it is 10 µg/mL with pure CEF against both gram-positive bacteria. This confirms the enhanced antibacterial activity of GO-PEG-CEF over pure CEF against gram-positive bacteria. These findings therefore show GO-PEG-CEF is promising as a sustained-release nanoantibiotic system for effective treatment against S. aureus and B. cereus infections.

Imprint cytology: a useful screening test for diagnosis of Helicobacter pylori in resource poor settings.

OBJECTIVE: The study aimed to compare the usefulness of two staining methods for imprint cytology for diagnosis of Helicobacter pylori infection. Gastric biopsy specimens (from dyspeptic patients attending routine upper gastrointestinal endoscopy) were placed on glass slides to obtain imprints. The imprints were stained with Toluidine blue and Giemsa stains separately and observed under ×400 magnification using a light microscope. Imprinted biopsies were sectioned and stained with H & E stain and Giemsa stain for histological analysis. Diagnosis of H. pylori infection in both imprint and histological sections were confirmed by a consultant pathologist. The sensitivity, specificity, positive predictive value and negative predictive value of each stain were calculated and benchmarked against histological diagnosis. RESULTS: Of the 55 dyspeptic patients enrolled in the study, 5 were positive for H. pylori by Toluidine blue stain and 4 by Giemsa stain. The sensitivity of Toluidine blue stain (57.1%) was higher than Giemsa stain (42.9%) while the specificity of both stains was equal (97.9%). Giemsa stain gave a better discrimination for identification of H. pylori bacteria among the mucosal background. Imprint cytology is a rapid, simple and cost effective diagnosis method that can supplement histological diagnosis.

Evaluation of the impact of six different DNA extraction methods for the representation of the microbial community associated with human chronic wound infections using a gel-based DNA profiling method.

Infected chronic wounds are polymicrobial in nature which include a diverse group of aerobic and anaerobic microorganisms. Majority of these communal microorganisms are difficult to grow in vitro. DNA fingerprinting methods such as polymerase chain reaction-denaturation gradient gel electrophoresis (PCR-DGGE) facilitate the microbial profiling of complex ecosystems including infected chronic wounds. Six different DNA extraction methods were compared for profiling of the microbial community associated with chronic wound infections using PCR-DGGE. Tissue debris obtained from chronic wound ulcers of ten patients were used for DNA extraction. Total nucleic acid was extracted from each specimen using six DNA extraction methods. The yield, purity and quality of DNA was measured and used for PCR amplification targeting V2-V3 region of eubacterial 16S rRNA gene. QIAGEN DNeasy Blood and Tissue Kit (K method) produced good quality genomic DNA compared to the other five DNA extraction methods and gave a broad diversity of bacterial communities in chronic wounds. Among the five conventional methods, bead beater/phenol-chloroform based DNA extraction method with STES buffer (BP1 method) gave a yield of DNA with a high purity and resulted in a higher DGGE band diversity. Although DNA extraction using heat and NaOH had the lowest purity, DGGE revealed a higher bacterial diversity. The findings suggest that the quality and the yield of genomic DNA are influenced by the DNA extraction protocol, thus a method should be carefully selected in profiling a complex microbial community.

Proinflammatory Cytokine IL-17 Shows a Significant Association with Helicobacter pylori Infection and Disease Severity.

BACKGROUND: The pro- and anti-inflammatory cytokines play an important role in the immune response against H. pylori infection. The proinflammatory cytokines of Th17 cells have been suggested to play a major role in H. pylori infection and resulting gastric inflammation. OBJECTIVE: The objective of this study was to compare the expression of selected inflammatory cytokines (IL-10, IL-17, IL-21, IL-23, and TNF-α) in H. pylori-infected patients and healthy controls and to understand their association with H. pylori infection and disease severity. RESULTS: The expression levels of IL-17 and IL-23 were significantly higher in H. pylori-infected patients. The expression of IL-21 was also higher in H. pylori-positive patients but there was no significant association with infection. IL-17 expression showed a significant increase with the severity of chronic gastritis. CONCLUSION: The proinflammatory cytokine, IL-17, shows a significant association with H. pylori infection and disease severity in a Sri Lankan dyspeptic patient population.