BioMed Research International: Infectious Diseases The latest articles from Hindawi Publishing Corporation © 2015 , Hindawi Publishing Corporation . All rights reserved. The Epidemiology and Economic Burden of Clostridium difficile Infection in Korea Mon, 02 Mar 2015 11:19:05 +0000 The prevalence of Clostridium difficile infection and the associated burden have recently increased in many countries. While the main risk factors for C. difficile infection include old age and antibiotic use, the prevalence of this infection is increasing in low-risk groups. These trends highlight the need for research on C. difficile infection. This study pointed out the prevalence and economic burden of C. difficile infection and uses the representative national data which is primarily from the database of the Korean Health Insurance Review and Assessment Service, for 2008–2011. The annual economic cost was measured using a prevalence approach, which sums the costs incurred to treat C. difficile infection. C. difficile infection prevalence was estimated to have increased from 1.43 per 100,000 in 2008 to 5.06 per 100,000 in 2011. Moreover, mortality increased from 69 cases in 2008 to 172 in 2011. The economic cost increased concurrently, from $2.4 million in 2008 to $7.6 million, $10.5 million, and $15.8 million in 2009, 2010, and 2011, respectively. The increasing economic burden of C. difficile infection over the course of the study period emphasizes the need for intervention to minimize the burden of a preventable illness like C. difficile infection. Hyung-Yun Choi, So-Youn Park, Young-Ae Kim, Tai-Young Yoon, Joong-Myung Choi, Bong-Keun Choe, So-Hee Ahn, Seok-Jun Yoon, Ye-Rin Lee, and In-Hwan Oh Copyright © 2015 Hyung-Yun Choi et al. All rights reserved. HIV-Associated Neurocognitive Disorders: The Relationship of HIV Infection with Physical and Social Comorbidities Sun, 01 Mar 2015 12:12:45 +0000 The prevalence of HIV (human immunodeficiency virus) associated neurocognitive disorders (HAND) will undoubtedly increase with the improved longevity of HIV-infected persons. HIV infection, itself, as well as multiple physiologic and psychosocial factors can contribute to cognitive impairment and neurologic complications. These comorbidities confound the diagnosis, assessment, and interventions for neurocognitive disorders. In this review, we discuss the role of several key comorbid factors that may contribute significantly to the development and progression of HIV-related neurocognitive impairment, as well as the current status of diagnostic strategies aimed at identifying HIV-infected individuals with impaired cognition and future research priorities and challenges. Ellen M. Tedaldi, Nancy L. Minniti, and Tracy Fischer Copyright © 2015 Ellen M. Tedaldi et al. All rights reserved. Analysis of Monocytic and Granulocytic Myeloid-Derived Suppressor Cells Subsets in Patients with Hepatitis C Virus Infection and Their Clinical Significance Sun, 01 Mar 2015 11:17:57 +0000 Myeloid-derived suppressor cells (MDSCs) have been shown to inhibit T-cell responses in many diseases, but, in hepatitis C virus (HCV) infected patients, MDSCs are still poorly studied. In this assay, we investigated the phenotype and frequency of two new populations of MDSCs denoted as monocytic and granulocytic MDSCs (M-MDSCs and G-MDSCs) in HCV infected patients and analyzed their clinical significance in these patients respectively. We found that the frequency of CD14+ cells (M-MDSCs) from HCV infected patients (mean ± SE, 3.134% ± 0.340%) was significantly increased when compared to healthy controls (mean ± SE, 1.764% ± 0.461%) (Z = −2.438, P = 0.015), while there was no statistical difference between the frequency of CD33+CD11b+CD15+ (G-MDSCs) of HCV infected patients and healthy donors (0.201% ± 0.038% versus 0.096% ± 0.026%, P > 0.05), which suggested that HCV infection could cause the proliferation of M-MDSCs instead of G-MDSCs. Besides, we found that the frequency of M-MDSCs in HCV infected patients had certain relevance with age (r = 0.358, P = 0.003); patients older than 40 years old group (mean ± SE, 3.673% ± 0.456%) had a significantly higher frequency of M-MDSCs than that of age less than 40 years old group (mean ± SE, 2.363% ± 0.482%) (Z = −2.685, P = 0.007). The frequency of M-MDSCs, however, had no correlation with HCV RNA loads, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and the level of liver inflammation degree. Gang Ning, Lanhui She, Lirong Lu, Ying Liu, Yingfu Zeng, Ying Yan, and Chaoshuang Lin Copyright © 2015 Gang Ning et al. All rights reserved. Clinical Usefulness of the 2010 Clinical and Laboratory Standards Institute Revised Breakpoints for Cephalosporin Use in the Treatment of Bacteremia Caused by Escherichia coli or Klebsiella spp. Sun, 22 Feb 2015 06:52:46 +0000 We investigated the clinical usefulness of the revised 2010 Clinical and Laboratory Standards Institute (CLSI) breakpoints for Escherichia coli and Klebsiella spp. Of 2,623 patients with bacteremia caused by E. coli or Klebsiella spp., 573 who had been treated appropriately with cephalosporin based on the CLSI 2009 guidelines were enrolled. There were no differences in the rates of treatment failure or mortality between the appropriately and inappropriately treated groups according to the CLSI 2010 guidelines. Additionally, in the matched case-control analysis, the treatment failure rate was higher in bacteremic patients with extended-spectrum β-lactamase- (ESBL-) producing but cephalosporin-susceptible organisms than in those with ESBL-nonproducing isolates when patients with urinary tract infections were excluded (44% and 0%, resp., ). In patients with bacteremia caused by E. coli or Klebsiella spp., the revised CLSI 2010 guidelines did not lead to poorer outcomes. However, ESBL production appeared to be associated with poor clinical outcomes in patients with bacteremia from sources other than the urinary tract. Nam Su Ku, Hae-Sun Chung, Jun Yong Choi, Dongeun Yong, Kyungwon Lee, June Myung Kim, and Yunsop Chong Copyright © 2015 Nam Su Ku et al. All rights reserved. Phytochemical Analysis and Modulation of Antibiotic Activity by Luehea paniculata Mart. & Zucc. (Malvaceae) in Multiresistant Clinical Isolates of Candida Spp. Mon, 16 Feb 2015 09:17:47 +0000 The high incidence of fungal infections has led to the continuous search for new drugs. Extracts of Luehea paniculata, a tree of multiple medicinal uses, were evaluated for anti-Candida activity, as well as its modulator potential of the Fluconazole antibiotic. Chemical prospecting of ethanol extracts of leaf and bark was carried out, the quantification of total phenols and flavonoids, characterized by the HPLC-DAD technique. The rosmarinic acid and the vitexin flavonoid were observed as major constituents in ELELP and ESWELP, respectively. Antioxidant activity was also evaluated by the method of scavenging the free radical DPPH, and quercetin was used as standard, obtaining IC50 values: 0.341 (mg/mL) for ELELP and 0.235 (mg/mL) for ESWELP. The microdilution assay was performed for antifungal activity against strains of Candida albicans, C. krusei, and C. tropicalis and showed minimum inhibitory concentrations values ≥1024 μg/mL. In the modulator action of extracts on Fluconazole against multiresistant clinical isolates of Candida (subinhibitory concentration minimum of 128 μg/mL), a significant synergism was observed, indicating that the extracts potentiated the antifungal effect against C. tropicalis, where antioxidant flavonoids could be responsible. This is the first report about modifying activity of the antibiotic action of a species of the genus Luehea. João T. Calixto Júnior, Selene M. Morais, Clécio G. Martins, Larissa G. Vieira, Maria Flaviana B. Morais-Braga, Joara N. P. Carneiro, Antonio J. P. Machado, Irwin R. A. Menezes, Saulo R. Tintino, and Henrique D. M. Coutinho Copyright © 2015 João T. Calixto Júnior et al. All rights reserved. Predictive Contribution of Neutrophil/Lymphocyte Ratio in Diagnosis of Brucellosis Tue, 03 Feb 2015 08:46:59 +0000 Here we wanted to investigate predictive value of neutrophil/lymphocyte ratio (NLR) and platelet/lymphocyte ratio (PLR) in the diagnosis of brucellosis. Thirty-two brucellosis patients diagnosed with positive serum agglutination test and thirty-two randomized healthy subjects were enrolled in this study retrospectively. Result with ROC analyzes the baseline NLR and hemoglobin values were found to be significantly associated with brucellosis (, , resp.). Herein we demonstrated for the first time that NLR values were significantly associated with brucellosis. This situation can help clinicians during diagnosis of brucellosis. Serdar Olt, Hasan Ergenç, and Seyyid Bilal Açıkgöz Copyright © 2015 Serdar Olt et al. All rights reserved. Research on Bacterial Virulence in the Developing Countries Mon, 02 Feb 2015 07:21:20 +0000 Angel Cataldi, Ana Lucia Nascimento, Armando Acosta, and Chensong Wan Copyright © 2015 Angel Cataldi et al. All rights reserved. Latent Tuberculosis Infection among a Large Cohort of Medical Students at a Teaching Hospital in Italy Sun, 01 Feb 2015 11:52:46 +0000 The surveillance of latent tuberculosis infection (LTBI) in both healthcare workers and healthcare students is considered fundamental for tuberculosis (TB) prevention. The aim of the present study was to estimate LTBI prevalence and evaluate potential risk-factors associated with this condition in a large cohort of medical students in Italy. In a cross-sectional study, performed between March and December 2012, 1511 eligible subjects attending the Medical School of the University of Genoa, trained at the IRCCS San Martino-IST Teaching Hospital of Genoa, were actively called to undergo the tuberculin skin test (TST). All the TST positive cases were confirmed with an interferon-gamma release assay (IGRA). A standardized questionnaire was collected for multivariate risk analysis. A total of 1302 (86.2%) students underwent TST testing and completed the questionnaire. Eleven subjects (0.8%) resulted TST positive and LTBI diagnosis was confirmed in 2 (0.1%) cases. Professional exposure to active TB patients (OR 21.7, 95% CI 2.9–160.2; value 0.003) and previous BCG immunization (OR 28.3, 95% CI 3.0–265.1; value 0.003) are independently associated with TST positivity. Despite the low prevalence of LTBI among Italian medical students, an occupational risk of TB infection still exists in countries with low circulation of Mycobacterium tuberculosis. Paolo Durando, Cristiano Alicino, Andrea Orsi, Ilaria Barberis, Chiara Paganino, Guglielmo Dini, Giovanni Mazzarello, Valerio Del Bono, Claudio Viscoli, Francesco Copello, Dimitri Sossai, Giovanni Orengo, Laura Sticchi, Filippo Ansaldi, and Giancarlo Icardi Copyright © 2015 Paolo Durando et al. All rights reserved. WHO Multidrug Therapy for Leprosy: Epidemiology of Default in Treatment in Agra District, Uttar Pradesh, India Sun, 01 Feb 2015 11:30:07 +0000 Aim. To study the magnitude of default, time of default, its causes, and final clinical outcome. Methods. Data collected in active surveys in Agra is analyzed. Patients were given treatment after medical confirmation and were followed up. The treatment default and other clinical outcomes were recorded. Results. Patients who defaulted have comparable demographic characteristics. However, among defaulters more women (62.7% in PB, 42.6% in MB) were seen than those in treatment completers (PB 52.7% and MB 35.9%). Nerve involvement was high in treatment completers: 45.7% in PB and 91.3% in MB leprosy. Overall default rate was lower (14.8%) in ROM than (28.8%) in standard MDT for PB leprosy (, ) and also for MB leprosy: 9.1% in ROM compared to 34.5% in MDT (, ). Default rate was not different (28.8% versus 34.5%, ) in both types of leprosy given MDT. Most patients defaulted at early stage of treatment and mainly due to manageable side effects. Conclusion. The default in standard MDT both for PB and MB leprosy was observed to be significantly higher than in ROM treatment. Most defaults occurred at early stage of treatment and major contribution of default is due to side effects like drowsiness, weakness, vomiting, diarrhea, and so forth, related to poor general health. Although about half of the defaulters were observed to be cured 2.2% in PB-MDT and 10.9% of MB-MDT developed disability. This is an issue due to default. Attempts are needed to increase treatment compliance. The use of specially designed disease related health education along with easily administered drug regimens may help to reduce default. Anil Kumar, Anita Girdhar, Joy Kumar Chakma, and Bhuwneswar Kumar Girdhar Copyright © 2015 Anil Kumar et al. All rights reserved. Impairment of Neutrophil Migration to Remote Inflammatory Site during Lung Histoplasmosis Thu, 29 Jan 2015 14:24:10 +0000 Histoplasma capsulatum (Hc) induces a pulmonary disease in which leukotrienes promote activation and recruitment of effectors cells. It is also well-recognized that leukotriene B4 (LTB4) and platelet-activating factor (PAF) induce leukocyte recruitment to inflammatory sites. We investigated the impact of pulmonary Hc infection on PMN migration to a remote inflammatory site. Our results show that pulmonary Hc infection impairs LTB4- or PAF-stimulated PMN recruitment to air pouch. Yet, remote inflammation did not modify PMN numbers in the bronchoalveolar lavage fluid (BALF) of Hc-infected mice. Interestingly, the concomitant administration of PAF and LTB4 receptor antagonists inhibited PMN recruitment to both BALF and the remote site, demonstrating cooperation between both mediators. Along that line, our results show that PAF-elicited PMN chemotaxis was abrogated in 5-lipoxygenase-deficient animals. These results suggest caution in the indiscriminate use of anti-inflammatory drugs during infectious diseases. Alexandra I. Medeiros, Adriana Secatto, Caroline Bélanger, Carlos A. Sorgi, Pierre Borgeat, Sylvie Marleau, and Lúcia H. Faccioli Copyright © 2015 Alexandra I. Medeiros et al. All rights reserved. High In Vitro Antibacterial Activity of Pac-525 against Porphyromonas gingivalis Biofilms Cultured on Titanium Thu, 29 Jan 2015 13:35:36 +0000 In order to investigate the potential of short antimicrobial peptides (AMPs) as alternative antibacterial agents during the treatment of peri-implantitis, the cytotoxic activity of three short AMPs, that is, Pac-525, KSL-W, and KSL, was determined using the MTT assay. The antimicrobial activity of these AMPs, ranging in concentration from 0.0039 mg/mL to 0.5 mg/mL, against the predominant planktonic pathogens, including Streptococcus sanguis, Fusobacterium nucleatum, and Porphyromonas gingivalis, involved in peri-implantitis was investigated. Furthermore, 2-day-old P. gingivalis biofilms cultured on titanium surfaces were treated with Pac-525 and subsequently observed and analysed using confocal laser scanning microscopy (CLSM). The average cell proliferation curve indicated that there was no cytotoxicity due to the three short AMPs. The minimum inhibitory concentration and minimum bactericidal concentration values of Pac-525 were 0.0625 mg/mL and 0.125 mg/mL, respectively, for P. gingivalis and 0.0078 mg/mL and 0.0156 mg/mL, respectively, for F. nucleatum. Using CLSM, we confirmed that compared to 0.1% chlorhexidine, 0.5 mg/mL of Pac-525 caused a significant decrease in biofilm thickness and a decline in the percentage of live bacteria. These data indicate that Pac-525 has unique properties that might make it suitable for the inhibition the growth of pathogenic bacteria around dental implants. Ji-yin Li, Xue-jin Wang, Li-na Wang, Xiao-xia Ying, Xiang Ren, Hui-ying Liu, Li Xu, and Guo-wu Ma Copyright © 2015 Ji-yin Li et al. All rights reserved. Pan-Genome Analysis of Human Gastric Pathogen H. pylori: Comparative Genomics and Pathogenomics Approaches to Identify Regions Associated with Pathogenicity and Prediction of Potential Core Therapeutic Targets Thu, 29 Jan 2015 06:54:04 +0000 Helicobacter pylori is a human gastric pathogen implicated as the major cause of peptic ulcer and second leading cause of gastric cancer (~70%) around the world. Conversely, an increased resistance to antibiotics and hindrances in the development of vaccines against H. pylori are observed. Pan-genome analyses of the global representative H. pylori isolates consisting of 39 complete genomes are presented in this paper. Phylogenetic analyses have revealed close relationships among geographically diverse strains of H. pylori. The conservation among these genomes was further analyzed by pan-genome approach; the predicted conserved gene families (1,193) constitute ~77% of the average H. pylori genome and 45% of the global gene repertoire of the species. Reverse vaccinology strategies have been adopted to identify and narrow down the potential core-immunogenic candidates. Total of 28 nonhost homolog proteins were characterized as universal therapeutic targets against H. pylori based on their functional annotation and protein-protein interaction. Finally, pathogenomics and genome plasticity analysis revealed 3 highly conserved and 2 highly variable putative pathogenicity islands in all of the H. pylori genomes been analyzed. Amjad Ali, Anam Naz, Siomar C Soares, Marriam Bakhtiar, Sandeep Tiwari, Syed S Hassan, Fazal Hanan, Rommel Ramos, Ulisses Pereira, Debmalya Barh, Henrique César Pereira Figueiredo, David W. Ussery, Anderson Miyoshi, Artur Silva, and Vasco Azevedo Copyright © 2015 Amjad Ali et al. All rights reserved. Oxidative Stress Control by Apicomplexan Parasites Wed, 28 Jan 2015 09:44:33 +0000 Apicomplexan parasites cause infectious diseases that are either a severe public health problem or an economic burden. In this paper we will shed light on how oxidative stress can influence the host-pathogen relationship by focusing on three major diseases: babesiosis, coccidiosis, and toxoplasmosis. Soraya S. Bosch, Thales Kronenberger, Kamila A. Meissner, Flávia M. Zimbres, Dirk Stegehake, Natália M. Izui, Isolmar Schettert, Eva Liebau, and Carsten Wrenger Copyright © 2015 Soraya S. Bosch et al. All rights reserved. Investigation of Ser315 Substitutions within katG Gene in Isoniazid-Resistant Clinical Isolates of Mycobacterium tuberculosis from South India Wed, 28 Jan 2015 09:44:30 +0000 Mutation at codon 315 of katG gene is the major cause for isoniazid (INH) resistance in Mycobacterium tuberculosis (M. tuberculosis). Substitution at codon 315 of katG gene was analyzed in 85 phenotypically resistant isolates collected from various parts of southern India by direct sequencing method. The obtained results were interpreted in the context of minimum inhibitory concentration (MIC) of INH. Of the 85 phenotypically resistant isolates, 56 (66%) were also correlated by the presence of resistance mutations in the katG gene; 47 of these isolates had ACC, 6 had AAC, 2 had ATC, and one had CGC codon. The frequency of Ser315 substitution in katG gene was found to be higher (70%) amongst multidrug-resistant (MDR) strains than among non-MDR (61%) INH-resistant isolates. Further, the frequency of mutations was found to be greater (74%) in isolates with higher MIC values in contrast to those isolates with low MIC values (58%). Therefore, the study identified high prevalence of Ser315Thr substitution in katG gene of INH-resistant isolates from south India. Also, isolates harboring this substitution were found to be associated with multidrug and high level INH resistance. A. Nusrath Unissa, N. Selvakumar, Sujatha Narayanan, C. Suganthi, and L. E. Hanna Copyright © 2015 A. Nusrath Unissa et al. All rights reserved. Clinical Epidemiology and Paraclinical Findings in Tuberculosis Patients in North of Iran Wed, 28 Jan 2015 06:39:44 +0000 Background. Mycobacterium tuberculosis (M.TB) causes a wide spectrum of clinical diseases. The prevalence of TB is different in various parts of Iran and throughout the world. The present study aimed to determine the clinical epidemiology and paraclinical findings of TB. Methods. A cross-sectional study was conducted from 2008 to 2013. Patient demographic, clinical, and radiologic characteristics, picked up from the TB patient’s files, were collected using a standard questionnaire format. Data was entered and analyzed using the SPSS version 16 statistical software and P value < 0.05 was considered statistically significant. Results. Out of 212 patients enrolled in this study 62% were male and the mean age was about 50 years old. 98.6% were Iranian, and 46.2% were rural. Prevalence of smear-positive TB was 66.4%. Prevalence of positive PPD was 50.7% with no significant difference between HIV-positive and -negative patients (P = 0.8). Prevalence of diabetes mellitus was 17%. 36% of the patients had history of smoking and about 29.3% were addicted to narcotics. Cough was the most common symptom (94.5%) and 84% had sputum. 15 cases (7%) had extrapulmonary TB. The mean time between the onset of symptoms and admission was 46.5 days. The delay for admission between urban and rural populations was not significantly different (P = 0.68); but for those who were in prison, the delay was significant (P = 0.02). About 46% of the patients had cavitary lesions in CXRs. Conclusion. Timely diagnosis of TB especially in prisoners by understanding its most important epidemiologic characteristics and clinical features can help to make an early treatment and prevent spread of mycobacteria and their complications. Farhang Babamahmoodi, Ahmad Alikhani, Jamshid Yazdani Charati, Amir Ghovvati, Fatemeh Ahangarkani, Leila Delavarian, and Abdolreza Babamahmoodi Copyright © 2015 Farhang Babamahmoodi et al. All rights reserved. Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Trichosporon asahii in Experimental and Clinical Samples Tue, 27 Jan 2015 08:43:07 +0000 Invasive trichosporonosis is a deep mycosis found mainly in immunocompromised hosts, and the major pathogen is Trichosporon asahii. We detected the species-specific intergenic spacers (IGS) of rRNA gene of T. asahii using a loop-mediated isothermal amplification (LAMP) assay in 15 isolates with 3 different visualization methods, including SYBR green detection, gel electrophoresis, and turbidimetric methods. The LAMP assay displayed superior rapidity to other traditional methods in the detection time; that is, only 1 h was needed for detection and identification of the pathogen DNA. Furthermore, the detection limit of the LAMP assay was more sensitive than the PCR assay. We also successfully detect the presence of T. asahii in samples from experimentally infected mice and samples from patients with invasive trichosporonosis caused by T. asahii, suggesting that this method may become useful in clinical applications in the near future. Jianfeng Zhou, Yong Liao, Haitao Li, Xuelian Lu, Xiufeng Han, Yanli Tian, Shanshan Chen, and Rongya Yang Copyright © 2015 Jianfeng Zhou et al. All rights reserved. Clinical Significance of Neutrophil Gelatinase-Associated Lipocalin in Crimean-Congo Hemorrhagic Fever Thu, 22 Jan 2015 13:28:46 +0000 Neutrophil gelatinase-associated lipocalin (NGAL), which is an important prognostic marker for sepsis and inflammatory diseases, is mostly released from neutrophils. Crimean-Congo hemorrhagic fever (CCHF) patients are generally neutropenic. We aimed to investigate whether there is a change in serum NGAL level and to investigate its effect on the recovery time (RT) during the course of CCHF. A total of 40 CCHF patients (19 females and 21 males) and 34 healthy controls (17 females and 17 males) were included in the study. The serum NGAL level and biochemical and hematological parameters were checked. The NGAL level of CCHF patients was significantly higher than that of the healthy controls (). A multivariate analysis showed that the independent prognostic factor for the prediction of the RT is the NGAL level (odds ratio [OR] 0.3, 95% confidence interval [Cl] 0.1–0.4, ). An elevated NGAL level was found to be associated with an increased RT in CCHF patients. The NGAL levels of CHHF patients might be elevated due to increased cytokine release, the presence of a tissue injury, and the release of immature neutrophils from the bone marrow into the peripheral stream. This may be a good prognostic factor in CHHF patients. Ayse Erturk, Erkan Cure, Emine Parlak, Medine Cumhur Cure, Serap Baydur Sahin, and Suleyman Yuce Copyright © 2015 Ayse Erturk et al. All rights reserved. The Diagnosis of Invasive and Noninvasive Pulmonary Aspergillosis by Serum and Bronchoalveolar Lavage Fluid Galactomannan Assay Thu, 22 Jan 2015 11:29:36 +0000 The incidence and mortality of invasive pulmonary aspergillosis (IPA) are rising, particularly in critically ill patients and patients with severe chronic obstructive pulmonary disease (COPD). Noninvasive aspergillosis occurring in these patients requires special attention because of the possibility of developing subsequent IPA, given the poor health and worsened immune state of these patients. We compared the performance of the Platelia galactomannan (GM) enzyme immunoassay in the bronchoalveolar lavage fluid (BALF) and serum. The sensitivity, and specificity of BALF-GM were 85.4% and 62.4%, and those of serum-GM were 67.9% and 93.5% at the cutoff index of 0.5. As the cutoff index increased, the specificity of BALF-GM detection was increased with the detriment of sensitivity. The area under the ROC curves was 0.817 (95% CI: 0.718–0.916) for BALF-GM and 0.819 (95% CI: 0.712–0.926) for serum-GM. The optimal cutoff index was 1.19 for BALF-GM, and the sensitivity and specificity were 67.9% and 89.2%. The BALF-GM assay is more sensitive in detecting pulmonary aspergillosis than serum-GM assay and fungal cultures. However, BALF-GM assay has a high false-positive rate at the cutoff index of 0.5. Hence, the diagnostic cutoff index of the BALF-GM assay should be improved to avoid the overdiagnosis of pulmonary aspergillosis in clinic. Shuzhen Zhang, Sibu Wang, Zhe Wan, Ruoyu Li, and Jin Yu Copyright © 2015 Shuzhen Zhang et al. All rights reserved. CD8+ T-Cells Count in Acute Myocardial Infarction in HIV Disease in a Predominantly Male Cohort Mon, 19 Jan 2015 12:07:35 +0000 Human Immunodeficiency Virus- (HIV-) infected persons have a higher risk for acute myocardial infarction (AMI) than HIV-uninfected persons. Earlier studies suggest that HIV viral load, CD4+ T-cell count, and antiretroviral therapy are associated with cardiovascular disease (CVD) risk. Whether CD8+ T-cell count is associated with CVD risk is not clear. We investigated the association between CD8+ T-cell count and incident AMI in a cohort of 73,398 people (of which 97.3% were men) enrolled in the U.S. Veterans Aging Cohort Study-Virtual Cohort (VACS-VC). Compared to uninfected people, HIV-infected people with high baseline CD8+ T-cell counts (>1065 cells/mm3) had increased AMI risk (adjusted , 95% CI: 1.46 to 2.28). There was evidence that the effect of CD8+ T-cell tertiles on AMI risk differed by CD4+ T-cell level: compared to uninfected people, HIV-infected people with CD4+ T-cell counts ≥200 cells/mm3 had increased AMI risk with high CD8+ T-cell count, while those with CD4+ T-cell counts <200 cells/mm3 had increased AMI risk with low CD8+ T-cell count. CD8+ T-cell counts may add additional AMI risk stratification information beyond that provided by CD4+ T-cell counts alone. Oluwatosin A. Badejo, Chung-Chou Chang, Kaku A. So-Armah, Russell P. Tracy, Jason V. Baker, David Rimland, Adeel A. Butt, Adam J. Gordon, Charles R. Rinaldo Jr., Kevin Kraemer, Jeffrey H. Samet, Hilary A. Tindle, Matthew B. Goetz, Maria C. Rodriguez-Barradas, Roger Bedimo, Cynthia L. Gibert, David A. Leaf, Lewis H. Kuller, Steven G. Deeks, Amy C. Justice, and Matthew S. Freiberg Copyright © 2015 Oluwatosin A. Badejo et al. All rights reserved. Assessment of Duration of Staying Free from Acquiring Rehappening Opportunistic Infections among Pre-ART People Living with HIV/AIDS between 2008 and 2013 Sun, 18 Jan 2015 12:59:30 +0000 Introduction. In regional state of the study area, HIV (Human Immunodeficiency Virus) prevalence is 2.2% and opportunistic infections (OIs) occurred in 88.9% of pre-ART (Antiretroviral Therapy) people living with HIV/AIDS (PLWHA). Even though OIs are prevalent in the study area, duration of staying free from acquiring rehappening opportunistic infections and its determinant factors are not studied. Method. The study was conducted in randomly selected 341 adult Pre-ART PLWHA who are included in chronic HIV care. OI free duration was estimated using the actuarial life table and Kaplan Meier survival. Cox proportional-hazard model was used to calculate hazard rate. Result. OIs were rediagnosed in three quarters (75.37%) participants. In each week the probability of getting new recurrence OI was about 15.04 per 1000 person weeks. The median duration of not acquiring OI recurrence was 54 weeks. After adjustment, variables associated with recurrence were employment status, marital status, exposure for prophylaxis and adherence to it, CD4 count, and hemoglobin value. Conclusion. Giving prophylaxis and counseling to adhere it, rise in CD4 and hemoglobin level, and enhancing job opportunities should be given for PLWHA who are on chronic HIV care while continuing the care. Habtamu Mellie Bizuayehu, Direslgne Misker Abyu, and Amlaku Mulat Aweke Copyright © 2015 Habtamu Mellie Bizuayehu et al. All rights reserved. Benzodiazepines and Z-Drug Use among HIV-Infected Patients in Taiwan: A 13-Year Nationwide Cohort Study Tue, 13 Jan 2015 11:03:00 +0000 Introduction. Benzodiazepines (BZDs) and zolpidem, zopiclone, and zaleplon (Z-drugs) are commonly prescribed to HIV-infected patients. We hypothesized that frequent BZD and Z-drug use among these patients may be associated with psychiatric illnesses, particularly in long-term users. Methods. We included 1,081 patients with HIV between 1998 and 2011 from the Taiwan National Health Insurance Research Database and matched them according to age, sex, and comorbidity with uninfected controls to investigate the psychiatric diagnoses and prescriptions of BZDs and Z-drugs. Cumulative defined daily dose (cDDD) was assessed as the indicator of the duration of medication exposure. Patients exhibiting a cDDD exceeding 180 were defined as long-term users. Results. The patients with HIV had an increased risk of any use (odds ratio (OR): 8.70, 95% confidence interval (CI): 6.82–10.97) and long-term use (OR: 5.06, 95% CI: 3.63–7.04) of BZD and Z-drugs compared with those without HIV during the follow-up after demographic data and psychiatric comorbidities were adjusted. Conclusion. A large proportion of the HIV-infected patients received prescriptions of BZDs and Z-drugs. Mood disorders, insomnia, anxiety disorders, HIV infection, and substance use disorder were substantial predictors among the BZD and Z-drug users. These findings suggest that providing psychiatric services for HIV-infected patients is vital. Han-Ting Wei, Mu-Hong Chen, Wing-Wai Wong, Yuan-Hwa Chou, Ying-Jay Liou, Tung-Ping Su, Tzeng-Ji Chen, and Ya-Mei Bai Copyright © 2015 Han-Ting Wei et al. All rights reserved. Parasites: From Source to Vector and Human Sun, 21 Dec 2014 06:11:27 +0000 Veeranoot Nissapatorn, Nongyao Sawangjaroen, Rogan Lee, and Subhash Chandra Parija Copyright © 2014 Veeranoot Nissapatorn et al. All rights reserved. Mycobacterium avium subsp. hominissuis Infection in Swine Associated with Peat Used for Bedding Mon, 15 Sep 2014 06:46:57 +0000 Mycobacterium avium subsp. hominissuis is an environmental bacterium causing opportunistic infections in swine, resulting in economic losses. Additionally, the zoonotic aspect of such infections is of concern. In the southeastern region of Norway in 2009 and 2010, an increase in condemnation of pig carcasses with tuberculous lesions was seen at the meat inspection. The use of peat as bedding in the herds was suspected to be a common factor, and a project examining pigs and environmental samples from the herds was initiated. Lesions detected at meat inspection in pigs originating from 15 herds were sampled. Environmental samples including peat from six of the herds and from three peat production facilities were additionally collected. Samples were analysed by culture and isolates genotyped by MLVA analysis. Mycobacterium avium subsp. hominissuis was detected in 35 out of 46 pigs, in 16 out of 20 samples of peat, and in one sample of sawdust. MLVA analysis demonstrated identical isolates from peat and pigs within the same farms. Polyclonal infection was demonstrated by analysis of multiple isolates from the same pig. To conclude, the increase in condemnation of porcine carcasses at slaughter due to mycobacteriosis seemed to be related to untreated peat used as bedding. Tone Bjordal Johansen, Angelika Agdestein, Bjørn Lium, Anne Jørgensen, and Berit Djønne Copyright © 2014 Tone Bjordal Johansen et al. All rights reserved. Cigarette Smoke-Exposed Candida albicans Increased Chitin Production and Modulated Human Fibroblast Cell Responses Thu, 11 Sep 2014 08:27:57 +0000 The predisposition of cigarette smokers for development of respiratory and oral bacterial infections is well documented. Cigarette smoke can also contribute to yeast infection. The aim of this study was to investigate the effect of cigarette smoke condensate (CSC) on C. albicans transition, chitin content, and response to environmental stress and to examine the interaction between CSC-pretreated C. albicans and normal human gingival fibroblasts. Following exposure to CSC, C. albicans transition from blastospore to hyphal form increased. CSC-pretreated yeast cells became significantly () sensitive to oxidation but significantly () resistant to both osmotic and heat stress. CSC-pretreated C. albicans expressed high levels of chitin, with 2- to 8-fold recorded under hyphal conditions. CSC-pretreated C. albicans adhered better to the gingival fibroblasts, proliferated almost three times more and adapted into hyphae, while the gingival fibroblasts recorded a significantly () slow growth rate but a significantly higher level of IL-1β when in contact with CSC-pretreated C. albicans. CSC was thus able to modulate both C. albicans transition through the cell wall chitin content and the interaction between C. albicans and normal human gingival fibroblasts. These findings may be relevant to fungal infections in the oral cavity in smokers. Humidah Alanazi, Abdelhabib Semlali, Laura Perraud, Witold Chmielewski, Andrew Zakrzewski, and Mahmoud Rouabhia Copyright © 2014 Humidah Alanazi et al. All rights reserved. Model of Break-Bone Fever via Beta-Derivatives Thu, 11 Sep 2014 06:16:02 +0000 Using the new derivative called beta-derivative, we modelled the well-known infectious disease called break-bone fever or the dengue fever. We presented the endemic equilibrium points under certain conditions of the physical parameters included in the model. We made use of an iteration method to solve the extended model. To show the efficiency of the method used, we have presented in detail the stability and the convergence of the method for solving the system (2). We presented the uniqueness of the special solution of system (2) and finally the numerical simulations were presented for various values of beta. Abdon Atangana and Suares Clovis Oukouomi Noutchie Copyright © 2014 Abdon Atangana and Suares Clovis Oukouomi Noutchie. All rights reserved. On the Mathematical Analysis of Ebola Hemorrhagic Fever: Deathly Infection Disease in West African Countries Thu, 11 Sep 2014 06:12:34 +0000 For a given West African country, we constructed a model describing the spread of the deathly disease called Ebola hemorrhagic fever. The model was first constructed using the classical derivative and then converted to the generalized version using the beta-derivative. We studied in detail the endemic equilibrium points and provided the Eigen values associated using the Jacobian method. We furthered our investigation by solving the model numerically using an iteration method. The simulations were done in terms of time and beta. The study showed that, for small portion of infected individuals, the whole country could die out in a very short period of time in case there is not good prevention. Abdon Atangana and Emile Franc Doungmo Goufo Copyright © 2014 Abdon Atangana and Emile Franc Doungmo Goufo. All rights reserved. Highlight on Advances in Nontuberculous Mycobacterial Disease in North America Thu, 11 Sep 2014 00:00:00 +0000 Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and exist as an important cause of pulmonary infections in humans. Pulmonary involvement is the most common disease manifestation of NTM and the incidence of NTM is growing in North America. Susceptibility to NTM infection is incompletely understood; therefore preventative tools are not well defined. Treatment of pulmonary nontuberculous mycobacterial (NTM) infection is difficult and entails multiple antibiotics and an extended treatment course. Also, there is a considerable variation in treatment management that should be considered before initiating treatment. We highlight the new findings in the epidemiology diagnosis and treatment of mycobacterial infections. We debate new advances regarding NTM infection in cystic fibrosis patients and solid organ transplant recipients. Finally, we introduce a new epidemiologic model for NTM disease based on virulence-exposure-host factors. Mehdi Mirsaeidi, Maham Farshidpour, Mary Beth Allen, Golnaz Ebrahimi, and Joseph O. Falkinham Copyright © 2014 Mehdi Mirsaeidi et al. All rights reserved. A Derived Network-Based Interferon-Related Signature of Human Macrophages Responding to Mycobacterium tuberculosis Sun, 31 Aug 2014 08:10:43 +0000 Network analysis of transcriptional signature typically relies on direct interaction between two highly expressed genes. However, this approach misses indirect and biological relevant interactions through a third factor (hub). Here we determine whether a hub-based network analysis can select an improved signature subset that correlates with a biological change in a stronger manner than the original signature. We have previously reported an interferon-related transcriptional signature (THP1r2Mtb-induced) from Mycobacterium tuberculosis (M. tb)-infected THP-1 human macrophage. We selected hub-connected THP1r2Mtb-induced genes into the refined network signature TMtb-iNet and grouped the excluded genes into the excluded signature TMtb-iEx. TMtb-iNet retained the enrichment of binding sites of interferon-related transcription factors and contained relatively more interferon-related interacting genes when compared to THP1r2Mtb-induced signature. TMtb-iNet correlated as strongly as THP1r2Mtb-induced signature on a public transcriptional dataset of patients with pulmonary tuberculosis (PTB). TMtb-iNet correlated more strongly in CD4+ and CD8+ T cells from PTB patients than THP1r2Mtb-induced signature and TMtb-iEx. When TMtb-iNet was applied to data during clinical therapy of tuberculosis, it resulted in the most pronounced response and the weakest correlation. Correlation on dataset from patients with AIDS or malaria was stronger for TMtb-iNet, indicating an involvement of TMtb-iNet in these chronic human infections. Collectively, the significance of this work is twofold: (1) we disseminate a hub-based approach in generating a biologically meaningful and clinically useful signature; (2) using this approach we introduce a new network-based signature and demonstrate its promising applications in understanding host responses to infections. Kang Wu, Hai Fang, Liang-Dong Lyu, Douglas B. Lowrie, Ka-Wing Wong, and Xiao-Yong Fan Copyright © 2014 Kang Wu et al. All rights reserved. Proteinases in Excretory-Secretory Products of Toxocara canis Second-Stage Larvae: Zymography and Modeling Insights Thu, 14 Aug 2014 06:49:17 +0000 Components released in excretory-secretory products of Toxocara canis larvae (TES) include phosphatidylethanolamine-binding proteins (TES26), mucins (TES120, MUC2-5), and C-type lectins (TES32, TES70) and their biochemical, immunological, and diagnostic properties have been extensively studied albeit proteinase activities towards physiological substrates are almost unknown. Proteolytic activities in TES samples were first analyzed by gel electrophoresis with gelatin as substrate. Major activities of ~400, 120, and 32 kDa in TES were relatively similar over a broad pH range (5.5–9.0) and all these were of the serine-type as leupeptin abolished gelatinolysis. Further, the ~400 kDa component degraded all physiological substrates tested (laminin, fibronectin, albumin, and goat IgG) and the 120 kDa component degraded albumin and goat IgG while proteinases of lower MW (45, 32, and 26 kDa) only degraded laminin and fibronectin, preferentially at alkaline pH (9.0). By protein modeling approaches using the known sequences of TES components, only TES26 and MUC4 displayed folding patterns significantly related to reference serine proteinases. These data suggest that most of serine proteinase activities secreted in vitro by infective larvae of T. canis have intriguing nature but otherwise help the parasite to affect multiple components of somatic organs and bodily fluids within the infected host. Gonzalo Ernesto González-Páez, Fernando Alba-Hurtado, Carlos Gerardo García-Tovar, and Raúl Argüello-García Copyright © 2014 Gonzalo Ernesto González-Páez et al. All rights reserved. Evaluation of Novel Tools to Facilitate the Detection and Characterization of Leprosy Patients in China Tue, 12 Aug 2014 08:57:53 +0000 Leprosy is the disabling outcome of chronic infection with Mycobacterium leprae. The disease often evades early detection, particularly now that fewer clinicians are able to confidently diagnose the disease following the integration of leprosy control measures within general health services in many countries. Although leprosy is officially eliminated in China, endemic regions remain in some difficult-to-reach, underdeveloped areas in Southwest China. In order to better understand the extent of M. leprae infection and identify new leprosy cases in a timely manner, simple tools that can detect infection and the early disease are required. In this report we evaluated the performance of antigen-specific ELISA, the NDO-LID rapid diagnostic test, and antigen-specific whole blood assays (WBA) as potential diagnostic tools. Our data support the use of antibody detection tests and WBA to facilitate the diagnosis of multibacillary and paucibacillary leprosy, respectively. These tools could be invaluable for increased, but simplified, monitoring of individuals in order to provide referrals for clinical exam and early leprosy diagnosis. Yan Wen, Yuan Gang You, Lian-Chao Yuan, You Hua Yuan, Ying Zhang, Malcolm S. Duthie, and Huan-Ying Li Copyright © 2014 Yan Wen et al. All rights reserved. Prevalence of Ischemic Heart Disease and Management of Coronary Risk in Daily Clinical Practice: Results from a Mediterranean Cohort of HIV-Infected Patients Thu, 07 Aug 2014 10:55:02 +0000 Background. There are conflicting data on the prevalence of coronary events and the quality of the management of modifiable cardiovascular risk factors (CVRF) in HIV-infected patients. Methods. We performed a retrospective descriptive study to determine the prevalence of coronary events and to evaluate the management of CVRF in a Mediterranean cohort of 3760 HIV-1-infected patients from April 1983 through June 2011. Results. We identified 81 patients with a history of a coronary event (prevalence 2.15%); 83% of them suffered an acute myocardial infarction. At the time of the coronary event, CVRF were highly prevalent (60.5% hypertension, 48% dyslipidemia, and 16% diabetes mellitus). Other CVRF, such as smoking, hypertension, lack of exercise, and body mass index, were not routinely assessed. After the coronary event, a significant decrease in total cholesterol and LDL-cholesterol was observed. However, the percentage of patients who maintained LDL-cholesterol > 100 mg/dL remained stable (from 46% to 41%, ). Patients using protease inhibitors associated with a favorable lipid profile increased over time . Conclusions. The prevalence of coronary events in our cohort is low. CVRF prevalence is high and their management is far from optimal. More aggressive interventions should be implemented to diminish cardiovascular risk in HIV-infected patients. Patricia Echeverría, Pere Domingo, Josep-María Llibre, Mar Gutierrez, Gracia Mateo, Jordi Puig, Anna Bonjoch, Nuria Pérez-Alvarez, Guillem Sirera, Bonaventura Clotet, and Eugenia Negredo Copyright © 2014 Patricia Echeverría et al. All rights reserved. Description of a Novel Adhesin of Mycobacterium avium Subsp. paratuberculosis Tue, 22 Jul 2014 08:13:46 +0000 The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP) by host cells are fibronectin (FN) dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu) as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the μM range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding. Mariana Noelia Viale, Gabriela Echeverria-Valencia, Pablo Romasanta, María Laura Mon, Marisa Fernandez, Emilio Malchiodi, María Isabel Romano, Andrea Karina Gioffré, and María de la Paz Santangelo Copyright © 2014 Mariana Noelia Viale et al. All rights reserved. Event-Specific Risk Factors Predicting Episodes of Unprotected Anal Intercourse with Male Nonregular Partners among Men Who Have Sex with Men Using Case-Crossover Study Design Sun, 20 Jul 2014 12:06:45 +0000 This study investigated event-specific factors that determine episodes of unprotected and protected anal intercourse (UAI and PAI) among 215 men who have sex with men (MSM), who used condoms inconsistently with nonregular partners (NRP) in the last six months, in Hong Kong. A case-crossover study design was used. Lower likelihood of episodes involving UAI with NRP was associated with (1) five partner attributes (NRP were <35 years old, at least three previous anal sex experiences with the NRP, perception that participant and the NRP had asymmetrical sexual experience, perception that the NRP was feminine, and liking toward the NRP; OR = 0.16–0.52), (2) six situational variables (the participant having had UAI with another man in the last week, having discussed condom use, perception that the NRP liked to use condom, partner’s suggestion to have PAI, participant’s suggestion to have PAI, and participant’s plan to use condoms; OR = 0.11–0.39), and (3) four environmental/setting variables (condoms already placed at the venue, display of condom use promotion materials, participant’s possession of a condom, and the NRP possessed a condom; OR = 0.27–0.45). HIV prevention targeting MSM should focus on event-specific protective factors, which may be different from those obtained from studies distinguishing condom users versus nonusers. Jinghua Li, Joseph T. F. Lau, Jing Gu, Chun Hao, and Coco H. Y. Lai Copyright © 2014 Jinghua Li et al. All rights reserved. Epidemiology, Phylogeny, and Evolution of Emerging Enteric Picobirnaviruses of Animal Origin and Their Relationship to Human Strains Thu, 17 Jul 2014 12:02:44 +0000 Picobirnavirus (PBV) which has been included in the list of viruses causing enteric infection in animals is highly versatile because of its broad host range and genetic diversity. PBVs are among the most recent and emerging small, nonenveloped viruses with a bisegmented double-stranded RNA genome, classified under a new family “Picobirnaviridae.” PBVs have also been detected from respiratory tract of pigs, but needs further close investigation for their inhabitant behavior. Though, accretion of genomic data of PBVs from different mammalian species resolved some of the ambiguity, quite a few questions and hypotheses regarding pathogenesis, persistence location, and evolution of PBVs remain unreciprocated. Evolutionary analysis reveals association of PBVs with partitiviruses especially fungi partitiviruses. Although, PBVs may have an ambiguous clinical implication, they do pose a potential public health concern in humans and control of PBVs mainly relies on nonvaccinal approach. Based upon the published data, from 1988 to date, generated from animal PBVs across the globe, this review provides information and discussion with respect to genetic analysis as well as evolution of PBVs of animal origin in relation to human strains. Yashpal S. Malik, Naveen Kumar, Kuldeep Sharma, Kuldeep Dhama, Muhammad Zubair Shabbir, Balasubramanian Ganesh, Nobumichi Kobayashi, and Krisztian Banyai Copyright © 2014 Yashpal S. Malik et al. All rights reserved. Computational Analysis of the Model Describing HIV Infection of CD4+T Cells Thu, 17 Jul 2014 00:00:00 +0000 An analysis of the model underpinning the description of the spread of HIV infection of CD4+T cells is examined in detail in this work. Investigations of the disease free and endemic equilibrium are done using the method of Jacobian matrix. An iteration technique, namely, the homotopy decomposition method (HDM), is implemented to give an approximate solution of nonlinear ordinary differential equation systems. The technique is described and illustrated with numerical examples. The approximated solution obtained via HDM is compared with those obtained via other methods to prove the trustworthiness of HDM. Moreover, the lessening and simplicity in calculations furnish HDM with a broader applicability. Abdon Atangana and Emile Franc Doungmo Goufo Copyright © 2014 Abdon Atangana and Emile Franc Doungmo Goufo. All rights reserved. Harnessing the Helminth Secretome for Therapeutic Immunomodulators Tue, 15 Jul 2014 09:19:29 +0000 Helminths are the largest and most complex pathogens to invade and live within the human body. Since they are not able to outpace the immune system by rapid antigen variation or faster cell division or retreat into protective niches not accessible to immune effector mechanisms, their long-term survival depends on influencing and regulating the immune responses away from the mode of action most damaging to them. Immunologists have focused on the excretory and secretory products that are released by the helminths, since they can change the host environment by modulating the immune system. Here we give a brief overview of the helminth-associated immune response and the currently available helminth secretome data. We introduce some major secretome-derived immunomodulatory molecules and describe their potential mode of action. Finally, the applicability of helminth-derived therapeutic proteins in the treatment of allergic and autoimmune inflammatory disease is discussed. Dana Ditgen, Emmanuela M. Anandarajah, Kamila A. Meissner, Norbert Brattig, Carsten Wrenger, and Eva Liebau Copyright © 2014 Dana Ditgen et al. All rights reserved. First Record of Isolation and Characterization of Methicillin Resistant Staphylococcus lugdunensis from Clinical Samples in Iraq Sun, 13 Jul 2014 11:08:32 +0000 This study was conducted to determine the frequency of Staphylococcus lugdunensis in different clinical samples. Out of 690 clinical samples, a total of 178 coagulase negative staphylococci (CoNS) isolates were recovered. CoNS were identified as 10 different species; 22 isolates belonged to Staphylococcus lugdunensis. Two specific genes for S. lugdunensis were used ( tanA gene and fbl gene) to confirm identification. Both of these specific genes were detected in 15 (68.1%) of 22 isolates that were identified phenotypically. The results of oxacillin MIC showed that 7 of the 15 (46.6%) S. lugdunensis isolates were oxacillin resistant. The antibiotic susceptibility testing against 16 antibiotics showed that resistance rates were variable towards these antibiotics. Eight of fifteen S. lugdunensis isolates (53.3%) were β-lactamase producer. Results of molecular detection of mecA gene found that mecA gene was detected in 6 (40%) of 15 S. lugdunensis. All of these 6 isolates (S1, S2, S3, S4, S5, and S6) were resistant to oxacillin. One isolate (S7) was resistant to oxacillin but mecA was not detected in this isolate. This study is a first record of isolation and characterization of methicillin resistant S. lugdunensis (MRSL) from clinical samples in Iraq. Alaa H. Al-Charrakh and Mohammed H. Obayes Copyright © 2014 Alaa H. Al-Charrakh and Mohammed H. Obayes. All rights reserved. Mycobacterium tuberculosis P-Type ATPases: Possible Targets for Drug or Vaccine Development Thu, 10 Jul 2014 11:38:46 +0000 Tuberculosis (TB) has been the biggest killer in the human history; currently, Mycobacterium tuberculosis (Mtb) kills nearly 2 million people each year worldwide. The high prevalence of TB obligates the identification of new therapeutic targets and the development of anti-TB vaccines that can control multidrug resistance and latent TB infections. Membrane proteins have recently been suggested as key targets for bacterial viability. Current studies have shown that mycobacteria P-type ATPases may play critical roles in ion homeostasis and in the response of mycobacteria to toxic substances in the intraphagosomal environment. In this review, we bring together the genomic, transcriptomic, and structural aspects of the P-type ATPases that are relevant during active and latent Mtb infections, which can be useful in determining the potential of these ATPases as drug targets and in uncovering their possible roles in the development of new anti-TB attenuated vaccines. Lorena Novoa-Aponte and Carlos Yesid Soto Ospina Copyright © 2014 Lorena Novoa-Aponte and Carlos Yesid Soto Ospina. All rights reserved. Modelling the Aggregation Process of Cellular Slime Mold by the Chemical Attraction Tue, 08 Jul 2014 08:54:41 +0000 We put into exercise a comparatively innovative analytical modus operandi, the homotopy decomposition method (HDM), for solving a system of nonlinear partial differential equations arising in an attractor one-dimensional Keller-Segel dynamics system. Numerical solutions are given and some properties show evidence of biologically practical reliance on the parameter values. The reliability of HDM and the reduction in computations give HDM a wider applicability. Abdon Atangana and P. D. Vermeulen Copyright © 2014 Abdon Atangana and P. D. Vermeulen. All rights reserved. Evaluation of Cocktails with Recombinant Proteins of Mycobacterium bovis for a Specific Diagnosis of Bovine Tuberculosis Tue, 08 Jul 2014 08:16:15 +0000 The Delayed type hypersensitivity skin test (DTH) and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB). The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B). In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1): ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2): ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria. María Laura Mon, Roberto Damián Moyano, Mariana Noelia Viale, María Alejandra Colombatti Olivieri, Ignacio José Gamietea, Valeria Noely Montenegro, Bernardo Alonso, María de la Paz Santangelo, Mahavir Singh, Rosario Duran, and María Isabel Romano Copyright © 2014 María Laura Mon et al. All rights reserved. Phylogenetic and In Silico Functional Analyses of Thermostable-Direct Hemolysin and tdh-Related Encoding Genes in Vibrio parahaemolyticus and Other Gram-Negative Bacteria Tue, 08 Jul 2014 06:54:25 +0000 Emergence and spread of pandemic strains of Vibrio parahaemolyticus have drawn attention to make detailed study on their genomes. The pathogenicity of V. parahaemolyticus has been associated with thermostable-direct hemolysin (TDH) and/or TDH-related hemolysin (TRH). The present study evaluated characteristics of tdh and trh genes, considering the phylogenetic and in silico functional features of V. parahaemolyticus and other bacteria. Fifty-two tdh and trh genes submitted to the GenBank were analyzed for sequence similarity. The promoter sequences of these genes were also analyzed from transcription start point to −35 regions and correlated with amino acid substitution within the coding regions. The phylogenetic analysis revealed that tdh and trh are highly distinct and also differ within the V. parahaemolyticus strains that were isolated from different geographical regions. Promoter sequence analysis revealed nucleotide substitutions and deletions at −18 and −19 positions among the pandemic, prepandemic, and nonpandemic tdh sequences. Many amino acid substitutions were also found within the signal peptide and also in the matured protein region of several TDH proteins as compared to TDH-S protein of pandemic V. parahaemolyticus. Experimental evidences are needed to recognize the importance of substitutions and deletions in the tdh and trh genes. Sushanta K. Bhowmik, Gururaja P. Pazhani, and Thandavarayan Ramamurthy Copyright © 2014 Sushanta K. Bhowmik et al. All rights reserved. Differential Expression of Immunogenic Proteins on Virulent Mycobacterium tuberculosis Clinical Isolates Mon, 07 Jul 2014 00:00:00 +0000 Molecular epidemiology has revealed that Mycobacterium tuberculosis (Mtb), formerly regarded as highly conserved species, displays a considerable degree of genetic variability that can influence the outcome of the disease as well as the innate and adaptive immune response. Recent studies have demonstrated that Mtb families found worldwide today differ in pathology, transmissibility, virulence, and development of immune response. By proteomic approaches seven proteins that were differentially expressed between a local clinical isolate from Latin-American-Mediterranean (LAM) and from Haarlem (H) lineages were identified. In order to analyze the immunogenic ability, recombinant Rv2241, Rv0009, Rv0407, and Rv2624c proteins were produced for testing specific antibody responses. We found that these proteins induced humoral immune responses in patients with drug-sensitive and drug-resistant tuberculosis with substantial cross-reactivity among the four proteins. Moreover, such reactivity was also correlated with anti-Mtb-cell surface IgM, but not with anti-ManLAM, anti-PPD, or anti-Mtb-surface IgG antibodies. Therefore, the present results describe new Mtb antigens with potential application as biomarkers of TB. Pablo Schierloh, Laura Klepp, Camila Vazquez, Roxana Valeria Rocha, Federico Carlos Blanco, Luciana Balboa, Beatriz López, Viviana Ritacco, Fabiana Bigi, and María del Carmen Sasiain Copyright © 2014 Pablo Schierloh et al. All rights reserved. Induction of Boosted Immune Response in Mice by Leptospiral Surface Proteins Expressed in Fusion with DnaK Sun, 06 Jul 2014 06:50:41 +0000 Leptospirosis is an important global disease of human and veterinary concern. Caused by pathogenic Leptospira, the illness was recently classified as an emerging infectious disease. Currently available veterinarian vaccines do not induce long-term protection against infection and do not provide cross-protective immunity. Several studies have suggested the use of DnaK as an antigen in vaccine formulation, due to an exceptional degree of immunogenicity. We focused on four surface proteins: rLIC10368 (Lsa21), rLIC10494, rLIC12690 (Lp95), and rLIC12730, previously shown to be involved in host-pathogen interactions. Our goal was to evaluate the immunogenicity of the proteins genetically fused with DnaK in animal model. The chosen genes were amplified by PCR methodology and cloned into pAE, an E. coli vector. The recombinant proteins were expressed alone or in fusion with DnaK at the N-terminus. Our results demonstrate that leptospiral proteins fused with DnaK have elicited an enhanced immune response in mice when compared to the effect promoted by the individual proteins. The boosted immune effect was demonstrated by the production of total IgG, lymphocyte proliferation, and significant amounts of IL-10 in supernatant of splenocyte cell cultures. We believe that this approach could be employed in vaccines to enhance presentation of antigens of Leptospira to professional immune cells. Marina V. Atzingen, Dunia Rodriguez, Gabriela Hase Siqueira, Luciana C. C. Leite, and Ana L. T. O. Nascimento Copyright © 2014 Marina V. Atzingen et al. All rights reserved. Low Prevalence of Hepatitis B and C Virus Markers among Children and Adolescents Tue, 01 Jul 2014 07:57:13 +0000 This study aimed to determine the prevalence of HBV and HCV among children and adolescents attending schools and daycare centres in Rio de Janeiro State, located in southern Brazil. Serum samples from 1,217 individuals aged 0 to 18 years were collected from 1999 to 2012 and tested for HBsAg, total anti-HBc, anti-HBs, and anti-HCV by ELISA. Reactive HBsAg and anti-HBc samples were tested for HBV DNA. Reactive anti-HCV samples were tested for HCV RNA and genotyped by RFLP. HBsAg was detected in 1.8% of individuals, and total anti-HBc was detected among 3.6% of individuals. Anti-HBs reactivity was found among 25.3% (322/1,217) of the individuals and increased from 6.28% in the years 1999-2000 to 76.2% in the years 2001–2012 . HBV DNA was detected in 18 of 51 individuals who presented with HBsAg or isolated anti-HBc, and nine were considered occult hepatitis B cases. Three individuals were anti-HCV- and HCV RNA-positive: two of them were infected with genotype 1, and the other was infected with genotype 3. Low levels of HBV and HCV markers were observed in children and adolescents. HBV immunity increased during the period of study, indicating that childhood universal HBV vaccination has been effective for controlling HBV infection in Brazil. Livia Melo Villar, Luciane Almeida Amado, Adilson José de Almeida, Vanessa Salete de Paula, Lia Laura Lewis-Ximenez, and Elisabeth Lampe Copyright © 2014 Livia Melo Villar et al. All rights reserved. Analysis of Antigens of Mycobacterium leprae by Interaction to Sera IgG, IgM, and IgA Response to Improve Diagnosis of Leprosy Sun, 29 Jun 2014 11:25:57 +0000 Till 2010, several countries have declared less than one leprosy patient among population of 10,000 and themselves feeling as eliminated from leprosy cases. However, new leprosy cases are still appearing from all these countries. In this situation one has to be confident to diagnose leprosy. This review paper highlighted already explored antigens for diagnosis purposes and finally suggested better combinations of protein antigens of M. leprae versus immunoglobulin as detector antibody to be useful for leprosy diagnosis. Avnish Kumar, Om Parkash, and Bhawneshwar K. Girdhar Copyright © 2014 Avnish Kumar et al. All rights reserved. Immunopathological Aspects of Experimental Trypanosoma cruzi Reinfections Tue, 24 Jun 2014 10:27:26 +0000 Chagas disease is caused by Trypanosoma cruzi infection. Besides the host-related factors, such as immune response and genetic background, the parasite, strain, and occurrences of reinfection episodes, may influence disease outcome. Our results demonstrate that both the primary infection and the reinfection with the Colombiana strain are connected with lower survival rate of the mice. After reinfection, parasitaemia is approximately ten times lower than in primary infected animals. Only Colombiana, Colombiana/Colombiana, and Y/Colombiana groups presented amastigote nests in cardiac tissue. Moreover, the mice infected and/or reinfected with the Colombiana strain had more T. cruzi nests, more intense inflammatory infiltrate, and higher in situ expression of TNF-α and IFN-γ than Y strain. Antigen-stimulated spleen cells from infected and/or reinfected animals produced higher levels of TNF-α, IFN-γ, and IL-10. Our results reinforce the idea that Chagas disease outcome is influenced by the strain of the infective parasite, being differentially modulated during reinfection episodes. It highlights the need of control strategies involving parasite strain characterization in endemic areas for Chagas disease. Juliana Reis Machado, Marcos Vinícius Silva, Diego Costa Borges, Crislaine Aparecida da Silva, Luis Eduardo Ramirez, Marlene Antônia dos Reis, Lúcio Roberto Castellano, Virmondes Rodrigues, and Denise Bertulucci Rocha Rodrigues Copyright © 2014 Juliana Reis Machado et al. All rights reserved. Helicobacter pylori Is Not Eradicated after Triple Therapy: A Nested PCR Based Study Mon, 23 Jun 2014 06:51:59 +0000 Detection of Helicobacter pylori after triple therapy is usually carried out by either rapid urease test (RUT), urea breath test (UBT), histology, bacterial isolation, and single round PCR or serological tests. In this study, antral biopsy specimens from 25 patients were tested for H. pylori by RUT, culture, histology, and nested PCR in their antral biopsy specimens before and after treatment. Three genes, namely, heat shock protein (hsp60), phosphoglucosamine mutase (ureC), and flagellar export ATP synthase (fliI) of H. pylori were targeted. Of the 25 antral biopsy specimens, the RUT, culture, histology, and nested PCR positivity dropped from 81.8% to 12%, 31% to 0%, 100 to 84%, and 100% to 92%, respectively, before and after therapy. Further, hsp60 specific amplicons from 23 out of 25 patients gave identical restriction pattern, while 6 fliI and 1 ureC specific amplicon produced different restriction pattern. Furthermore, variations in fliI gene sequences in H. pylori after treatment were also confirmed by sequencing and compared in silico. Nested PCR based detection of H. pylori is more sensitive method to detect H. pylori after therapy than culture, RUT, and histology. Further, this study suggests that H. pylori is not eradicated completely after triple therapy. Saurabh Kumar Patel, Girish Narayan Mishra, Chandra Bhan Pratap, Ashok Kumar Jain, and Gopal Nath Copyright © 2014 Saurabh Kumar Patel et al. All rights reserved. Regional Warming and Emerging Vector-Borne Zoonotic Dirofilariosis in the Russian Federation, Ukraine, and Other Post-Soviet States from 1981 to 2011 and Projection by 2030 Thu, 19 Jun 2014 09:15:01 +0000 We analyze through a climatic model the influence of regional warming on the geographical spreading and potential risk of infection of human dirofilariosis in Russia, Ukraine, and other post-Soviet states from 1981 to 2011 and estimate the situation by 2030. The model correctly predicts the spatiotemporal location of 97.10% of 2154 clinical cases reported in the area during the studied period, identified by a retrospective review of the literature. There exists also a significant correlation between annual predicted Dirofilaria generations and calculated morbidity. The model states the progressive increase of 14.8% in the potential transmission area, up to latitude 64°N, and 14.7% in population exposure. By 2030 an increase of 18.5% in transmission area and 10.8% in population exposure is expected. These findings strongly suggest the influence of global warming in both geographical spreading and increase in the number of Dirofilaria generations. The results should alert about the epidemiological behavior of dirofilariosis and other mosquito-borne diseases in these and other countries with similar climatic characteristics. Vladimir Kartashev, Alexandr Afonin, Javier González-Miguel, Rosa Sepúlveda, Luis Simón, Rodrigo Morchón, and Fernando Simón Copyright © 2014 Vladimir Kartashev et al. All rights reserved. Seroprevalence of Antibodies against Pkn1, a Novel Potential Immunogen, in Chlamydia trachomatis-Infected Macaca nemestrina and Human Patients Wed, 18 Jun 2014 08:26:51 +0000 Chlamydia trachomatis (CT) is an important cause of sexually transmitted genital tract infections (STIs) and trachoma. Despite major research into chlamydial pathogenesis and host immune responses, immunoprotection has been hampered by the incomplete understanding of protective immunity in the genital tract. Characterized vaccine candidates have shown variable efficacy ranging from no protection to partial protection in vivo. It is therefore a research priority to identify novel chlamydial antigens that may elicit protective immune responses against CT infection. In the present study we assessed the seroprevalence of antibodies against protein kinase1 (Pkn1), DNA ligaseA (LigA), and major outer membrane protein A (OmpA) following natural CT infection in humans and in experimentally induced CT infection in Macaca nemestrina. Antigenic stretches of Pkn1, LigA, and OmpA were identified using bioinformatic tools. Pkn1, LigA, and OmpA genes were cloned in bacterial expression vector and purified by affinity chromatography. Our results demonstrate significantly high seroprevalence of antibodies against purified Pkn1 and OmpA in sera obtained from the macaque animal model and human patients infected with CT. In contrast no significant seroreactivity was observed for LigA. The seroprevalence of antibodies against Pkn1 suggest that nonsurface chlamydial proteins could also be important for developing vaccines for C. trachomatis. Achchhe L. Patel, Prashant K. Mishra, Divya Sachdev, Uma Chaudhary, Dorothy L. Patton, and Daman Saluja Copyright © 2014 Achchhe L. Patel et al. All rights reserved. Liposomal Formulation of Turmerone-Rich Hexane Fractions from Curcuma longa Enhances Their Antileishmanial Activity Wed, 18 Jun 2014 00:00:00 +0000 Promastigote forms of Leishmania amazonensis were treated with different concentrations of two fractions of Curcuma longa cortex rich in turmerones and their respective liposomal formulations in order to evaluate growth inhibition and the minimal inhibitory concentration (MIC). In addition, cellular alterations of treated promastigotes were investigated under transmission and scanning electron microscopies. LipoRHIC and LipoRHIWC presented lower MIC, 5.5 and 12.5 μg/mL, when compared to nonencapsulated fractions (125 and 250 μg/mL), respectively, and to ar-turmerone (50 μg/mL). Parasite growth inhibition was demonstrated to be dose-dependent. Important morphological changes as rounded body and presence of several roles on plasmatic membrane could be seen on L. amazonensis promastigotes after treatment with subinhibitory concentration (2.75 μg/mL) of the most active LipoRHIC. In that sense, the hexane fraction from the turmeric cortex of Curcuma longa incorporated in liposomal formulation (LipoRHIC) could represent good strategy for the development of new antileishmanial agent. Ana Claudia F. Amaral, Luciana A. Gomes, Jefferson Rocha de A. Silva, José Luiz P. Ferreira, Aline de S. Ramos, Maria do Socorro S. Rosa, Alane B. Vermelho, and Igor A. Rodrigues Copyright © 2014 Ana Claudia F. Amaral et al. All rights reserved. High Prevalence of Panton-Valentine Leukocidin (PVL) Genes in Nosocomial-Acquired Staphylococcus aureus Isolated from Tertiary Care Hospitals in Nepal Wed, 18 Jun 2014 00:00:00 +0000 Methicillin-resistant Staphylococcus aureus (MRSA) carrying the important virulence determinant, Panton-Valentine leukocidin (PVL), is an emerging infectious pathogen associated with skin and soft tissue infections as well as life-threatening invasive diseases. In carrying out the first PVL prevalence study in Nepal, we screened 73 nosocomial isolates of S. aureus from 2 tertiary care Nepali hospitals and obtained an overall PVL-positivity rate of 35.6% among the hospital isolates: 26.1% of MRSA and 51.9% of methicillin sensitive S. aureus (MSSA) isolates were found to be positive for the PVL genes. PVL prevalence was not associated with a specific (i) infection site, (ii) age group, or (iii) hospital of origin. It was found to be positively associated with heterogeneous MRSA (73.3%) compared to homogeneous MRSA (3.2%) and MSSA (51.9%); negatively associated with multiresistant MRSA (22%) compared to nonmultiresistant MRSA (60%) and MSSA (51.9%); and positively associated with macrolide-streptogramin B resistance (93.8%) compared to macrolide-lincosamide-streptogramin B resistance (0%) and no-resistance (45.8%) types. Macrolide-streptogramin B resistance was confirmed by the presence of msr(A) gene. Restriction pattern analyses provided evidence to support the circulation of a limited number of clones of PVL-positive MRSA, arguing for the adaptability of these isolates to a hospital setting. Bidya Shrestha, Winny Singh, V. Samuel Raj, Bharat Mani Pokhrel, and Tribhuban Mohan Mohapatra Copyright © 2014 Bidya Shrestha et al. All rights reserved. Performance and Logistical Challenges of Alternative HIV-1 Virological Monitoring Options in a Clinical Setting of Harare, Zimbabwe Sun, 15 Jun 2014 05:25:06 +0000 We evaluated a low-cost virological failure assay (VFA) on plasma and dried blood spot (DBS) specimens from HIV-1 infected patients attending an HIV clinic in Harare. The results were compared to the performance of the ultrasensitive heat-denatured p24 assay (p24). The COBAS AmpliPrep/COBAS TaqMan HIV-1 test, version 2.0, served as the gold standard. Using a cutoff of 5,000 copies/mL, the plasma VFA had a sensitivity of 94.5% and specificity of 92.7% and was largely superior to the VFA on DBS (sensitivity = 61.9%; specificity = 99.0%) or to the p24 (sensitivity = 54.3%; specificity = 82.3%) when tested on 302 HIV treated and untreated patients. However, among the 202 long-term ART-exposed patients, the sensitivity of the VFA decreased to 72.7% and to 35.7% using a threshold of 5,000 and 1,000 RNA copies/mL, respectively. We show that the VFA (either on plasma or on DBS) and the p24 are not reliable to monitor long-term treated, HIV-1 infected patients. Moreover, achieving acceptable assay sensitivity using DBS proved technically difficult in a less-experienced laboratory. Importantly, the high level of virological suppression (93%) indicated that quality care focused on treatment adherence limits virological failure even when PCR-based viral load monitoring is not available. Pascale Ondoa, Tinei Shamu, Michelle Bronze, Maureen Wellington, Tamara Sonia Boender, Corry Manting, Kim Steegen, Rudi Luethy, and Tobias Rinke de Wit Copyright © 2014 Pascale Ondoa et al. All rights reserved. Directly-Observed Intermittent Therapy versus Unsupervised Daily Regimen during the Intensive Phase of Antituberculosis Therapy in HIV Infected Patients Wed, 11 Jun 2014 14:12:22 +0000 The World Health Organization strongly recommends using daily antituberculosis therapy (ATT) during the intensive phase for HIV infected patients. India has the highest burden of tuberculosis in the world, but HIV infected patients are still receiving intermittent ATT. In this study we compared the mortality in patients who received directly-observed intermittent ATT versus self-administered daily ATT with fixed dose combinations during the intensive phase in a context of freely available antiretroviral therapy. The study included 1460 patients, 343 in the intermittent ATT group and 1117 in the daily ATT group. Baseline covariates of the two groups were balanced using inverse probability of treatment weighting based on propensity score methods. In a sensitivity analysis, continuous variables (albumin, CD4 count, and age) were modelled using restricted cubic smoothing splines. Compared with patients who received daily ATT, patients who received intermittent ATT had a 40% higher risk of mortality (1.4 hazard ratio; 95% confidence interval, 1.14–1.7). We estimated that the use of daily ATT could achieve a 10% absolute reduction in mortality at 12 months. Self-administered daily ATT was not associated with an increased risk of default from treatment. These results support the immediate implementation of daily ATT for HIV infected patients during the intensive phase in India. Gerardo Alvarez-Uria, Manoranjan Midde, Raghavakalyan Pakam, and Praveen Kumar Naik Copyright © 2014 Gerardo Alvarez-Uria et al. All rights reserved. Exposure of a 23F Serotype Strain of Streptococcus pneumoniae to Cigarette Smoke Condensate Is Associated with Selective Upregulation of Genes Encoding the Two-Component Regulatory System 11 (TCS11) Wed, 11 Jun 2014 12:55:49 +0000 Alterations in whole genome expression profiles following exposure of the pneumococcus (strain 172, serotype 23F) to cigarette smoke condensate (160 μg/mL) for 15 and 60 min have been determined using the TIGR4 DNA microarray chip. Exposure to CSC resulted in the significant (–0.0006) upregulation of the genes encoding the two-component regulatory system 11 (TCS11), consisting of the sensor kinase, hk11, and its cognate response regulator, rr11, in the setting of increased biofilm formation. These effects of cigarette smoke on the pneumococcus may contribute to colonization of the airways by this microbial pathogen. Riana Cockeran, Jenny A. Herbert, Timothy J. Mitchell, Thérèse Dix-Peek, Caroline Dickens, Ronald Anderson, and Charles Feldman Copyright © 2014 Riana Cockeran et al. All rights reserved. Characterization of Shiga Toxin-Producing Escherichia coli Isolated from Ground Beef Collected in Different Socioeconomic Strata Markets in Buenos Aires, Argentina Mon, 09 Jun 2014 09:01:17 +0000 Consumption of raw/undercooked ground beef is the most common route of transmission of Shiga toxin-producing E. coli (STEC). The aim of the study was to determine the STEC contamination level of the ground beef samples collected in 36 markets of different socioeconomic strata in Buenos Aires, Argentina, and the characterization of the isolated strains. Ninety-one out of 252 (36.1%) samples were stx+. Fifty-seven STEC strains were recovered. Eleven STEC strains belonged to O157 serogroup, and 46 to non-O157 serogroups. Virulence markers of the 57 STEC were stx1, 5.3% (3/57); stx2, 86.0% (49/57); stx1/stx2, 8.8% (5/57); ehxA, 61.4% (35/57); eae, 26.3% (15/57); saa, 24.6% (14/57). Shiga toxin subtypes were stx2, 31.5% (17/54); stx2c-vhb, 24.1% (13/54); stx2c-vha, 20.4% (11/54); stx2/stx2c-vha, 14.8% (8/54); stx2/stx2c-vhb, 5.6% (3/54); stx2c-vha/vhb, 3.7% (2/54). Serotypes O178:H19 and O157:H7 were prevalent. Contamination rate of STEC in all strata was high, and the highest O157 contamination was observed at low strata at several sampling rounds. Persistence of STEC was not detected. Sixteen strains (28.1%) were resistant to ampicillin, streptomycin, amikacin, or tetracycline. The STEC contamination level of ground beef could vary according to the sociocultural characteristics of the population. Patricia Llorente, Laura Barnech, Kinue Irino, María Valeria Rumi, and Adriana Bentancor Copyright © 2014 Patricia Llorente et al. All rights reserved. Recombinant Dense Granular Protein (GRA5) for Detection of Human Toxoplasmosis by Western Blot Thu, 29 May 2014 11:55:21 +0000 Toxoplasma gondii infects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of whole Toxoplasma lysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressed T. gondii dense granular protein-5 (GRA5) in Escherichia coli and isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronic T. gondii infections (sensitivities of 46.8% and 61.2%, resp.). Xiao Teng Ching, Yee Ling Lau, Mun Yik Fong, Veeranoot Nissapatorn, and Hemah Andiappan Copyright © 2014 Xiao Teng Ching et al. All rights reserved. Added Value of QuantiFERON TB-Gold in-Tube for Detecting Latent Tuberculosis Infection among Persons Living with HIV/AIDS Thu, 29 May 2014 09:34:36 +0000 Objective. To evaluate the added value of QuantiFERON TB-Gold in-Tube (QTF-GIT) over the tuberculin skin testing (TST) for detecting latent tuberculosis (TB) infection (LTBI) among patients with AIDS in a city with a low TB incidence rate (11.1/100,000 inhabitants) and universal BCG coverage. Methods. Three hundred consecutive patients with AIDS in eight outpatient sexually transmitted disease public clinics in Brasilia were submitted to QFT-IT and TST between May 2011 and March 2013. A positive result of either test was considered to be LTBI. Results. Median CD4-cell count was 477.5 cells/mm3; 295 (98.3%) were using antiretroviral therapy. Eighteen patients (6%, 95% CI: 3.6%–9.3%) had LTBI, of whom 4 (1.3%, 95% CI: 0.04%–2.63%) had only a positive TST, 8 (2.7%, 95% CI: 0.8%–4.5%) had only a QFT-GIT positive test, and 6 (2%, 95% CI: 0.4%–3.6%) had positive results for both tests. This represents an 81.8% relative increase in LTBI detection when QFT-GIT is added to TST. The concordance between both tests was 96% (). Conclusions. The QFT-GIT alone was more effective to detect LTBI than TST alone and had an 81% added value as an add-on sequential test in this population with mild immunosuppression. The cost-effectiveness of these strategies remains to be evaluated. Josiane Maria Oliveira Souza, Maria do Socorro Nantua Evangelista, and Anete Trajman Copyright © 2014 Josiane Maria Oliveira Souza et al. All rights reserved. HS1,2 Ig Enhancer Alleles Association to AIDS Progression in a Pediatric Cohort Infected with a Monophyletic HIV-Strain Tue, 20 May 2014 12:53:20 +0000 Alteration in the humoral immune response has been observed during HIV infection. The polymorphisms of enhancer HS1,2, member of the 3′ regulatory region of the Ig heavy chain cluster, may play a role in the variation of the humoral response leading to pathological conditions. To assess the role of the HS1,2 polymorphic variants in the progression of AIDS, the HS1,2-A allelic frequencies were investigated in a cohort of HIV infected pediatric subjects from a nosocomial outbreak with a monophyletic strain of HIV. From a total group of 418 HIV infected children in the outbreak cohort, 42 nonprogressors and 31 progressors without bias due to antiretroviral therapy were evaluated. HS1,2 allele *1 has been associated with nonprogressors (allelic frequency: 51.19% versus 33.87% in progressors, OR 0.5, and , while allele *2 has been associated with progression (allelic frequency: 48.39% versus 30.95% in nonprogressors, OR 2.1, and . Further, only subjects carrying allele *2 in absence of allele *1, either in homozygous condition for allele *2 [nonprogressors 2/42 (4.76%), Progressors 7/31 (22.58%), OR 5.8, and or in combination with other allelic variants [nonprogressors 7/42 (16.67%), Progressors 13/31 (41.93%), OR 3.61, and , have been associated with HIV progression to AIDS. In conclusion, while the HS1,2 allele *1 has a protective effect on HIV progression when present, allele *2 is associated with progression toward AIDS when allele *1 is absent. Carla Montesano, Vincenzo Giambra, Domenico Frezza, Paolo Palma, Eliseo Serone, Guido Castelli Gattinara, Maurizio Mattei, Giorgio Mancino, Vittorio Colizzi, and Massimo Amicosante Copyright © 2014 Carla Montesano et al. All rights reserved. Characterization of a Mycobacterium avium subsp. avium Operon Associated with Virulence and Drug Detoxification Tue, 20 May 2014 00:00:00 +0000 The lprG-p55 operon of Mycobacterium tuberculosis and Mycobacterium bovis is involved in the transport of toxic compounds. P55 is an efflux pump that provides resistance to several drugs, while LprG is a lipoprotein that modulates the host's immune response against mycobacteria. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in mice and increases susceptibility to toxic compounds. In order to gain insight into the function of LprG in the Mycobacterium avium complex, in this study, we assayed the effect of the deletion of lprG gene in the D4ER strain of Mycobacterium avium subsp. avium. The replacement of lprG gene with a hygromycin cassette caused a polar effect on the expression of p55. Also, a twofold decrease in ethidium bromide susceptibility was observed and the resistance to the antibiotics rifampicin, amikacin, linezolid, and rifabutin was impaired in the mutant strain. In addition, the mutation decreased the virulence of the bacteria in macrophages in vitro and in a mice model in vivo. These findings clearly indicate that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of MAA in vitro and in vivo. Mariana Noelia Viale, Kun Taek Park, Belén Imperiale, Andrea Karina Gioffre, María Alejandra Colombatti Olivieri, Roberto Damián Moyano, Nora Morcillo, María de la Paz Santangelo, William Davis, and María Isabel Romano Copyright © 2014 Mariana Noelia Viale et al. All rights reserved. Differential Macrophage Response to Slow- and Fast-Growing Pathogenic Mycobacteria Sun, 18 May 2014 12:50:16 +0000 Nontuberculous mycobacteria (NTM) have recently been recognized as important species that cause disease even in immunocompetent individuals. The mechanisms that these species use to infect and persist inside macrophages are not well characterised. To gain insight concerning this process we used THP-1 macrophages infected with M. abscessus, M. fortuitum, M. celatum, and M. tuberculosis. Our results showed that slow-growing mycobacteria gained entrance into these cells with more efficiency than fast-growing mycobacteria. We have also demonstrated that viable slow-growing M. celatum persisted inside macrophages without causing cell damage and without inducing reactive oxygen species (ROS), as M. tuberculosis caused. In contrast, fast-growing mycobacteria destroyed the cells and induced high levels of ROS. Additionally, the macrophage cytokine pattern induced by M. celatum was different from the one induced by either M. tuberculosis or fast-growing mycobacteria. Our results also suggest that, in some cases, the intracellular survival of mycobacteria and the immune response that they induce in macrophages could be related to their growth rate. In addition, the modulation of macrophage cytokine production, caused by M. celatum, might be a novel immune-evasion strategy used to survive inside macrophages that is different from the one reported for M. tuberculosis. A. Cecilia Helguera-Repetto, Rommel Chacon-Salinas, Jorge F. Cerna-Cortes, Sandra Rivera-Gutierrez, Vianney Ortiz-Navarrete, Iris Estrada-Garcia, and Jorge A. Gonzalez-y-Merchand Copyright © 2014 A. Cecilia Helguera-Repetto et al. All rights reserved. Microbubble-Mediated Ultrasound Enhances the Lethal Effect of Gentamicin on Planktonic Escherichia coli Thu, 15 May 2014 14:21:59 +0000 Previous research has found that low-intensity ultrasound enhanced the lethal effect of gentamicin on planktonic E. coli. We aimed to further investigate whether microbubble-mediated low-intensity ultrasound could further enhance the antimicrobial efficacy of gentamicin. The planktonic E. coli (ATCC 25922) was distributed to four different interventions: control (), microbubble only (), ultrasound only (), and microbubble-mediated ultrasound (). Ultrasound was applied with 100 mW/cm2 (average intensity) and 46.5 KHz, which presented no bactericidal activity. After 12 h, plate counting was used to estimate the number of bacteria, and bacterial micromorphology was observed with transmission electron microscope. The results showed that the viable counts of E. coli in were decreased by 1.01 to 1.42 log10 CFU/mL compared with (). The minimal inhibitory concentration (MIC) of gentamicin against E. coli was 1 μg/mL in the and groups, lower than that in the and groups (2 μg/mL). Transmission electron microscopy (TEM) images exhibited more destruction and higher thickness of bacterial cell membranes in the than those in other groups. The reason might be the increased permeability of cell membranes for gentamicin caused by acoustic cavitation. Han-Xiao Zhu, Xun-Zi Cai, Zhong-Li Shi, Bin Hu, and Shi-Gui Yan Copyright © 2014 Han-Xiao Zhu et al. All rights reserved. Chagas’ Disease: Pregnancy and Congenital Transmission Thu, 15 May 2014 07:40:07 +0000 Chagas disease is a chronic infection that kills approximately 12,000 people a year. Mass migration of chronically infected and asymptomatic persons has caused globalization of Chagas disease and has made nonvectorial infection, including vertical and blood-borne transmission, more of a threat to human communities than vectorial infection. To control transmission, it is essential to test all pregnant women living in endemic countries and all pregnant women having migrated from, or having lived in, endemic countries. All children born to seropositive mothers should be tested not only within the first month of life but also at ~6 months and ~12 months of age. The diagnosis is made by identification of the parasite in blood before the age of 6 months and by identification of the parasite in blood and/or positive serology after 10 months of age. Follow up for a year is essential as a significant proportion of cases are initially negative and are only detected at a later stage. If the condition is diagnosed and treated early, the clinical response is excellent and the majority of cases are cured. Ana María Cevallos and Roberto Hernández Copyright © 2014 Ana María Cevallos and Roberto Hernández. All rights reserved. Immunization with the Recombinant Cholera Toxin B Fused to Fimbria 2 Protein Protects against Bordetella pertussis Infection Tue, 13 May 2014 12:01:58 +0000 This study examined the immunogenic properties of the fusion protein fimbria 2 of Bordetella pertussis (Fim2)—cholera toxin B subunit (CTB) in the intranasal murine model of infection. To this end B. pertussis Fim2 coding sequence was cloned downstream of the cholera toxin B subunit coding sequence. The expression and assembly of the fusion protein into pentameric structures (CTB-Fim2) were evaluated by SDS-PAGE and monosialotetrahexosylgaglioside (GM1-ganglioside) enzyme-linked immunosorbent assay (ELISA). To evaluate the protective capacity of CTB-Fim2, an intraperitoneal or intranasal mouse immunization schedule was performed with 50 μg of CTB-Fim2. Recombinant (rFim2) or purified (BpFim2) Fim2, CTB, and phosphate-buffered saline (PBS) were used as controls. The results showed that mice immunized with BpFim2 or CTB-Fim2 intraperitoneally or intranasally presented a significant reduction in bacterial lung counts compared to control groups ( or resp.). Moreover, intranasal immunization with CTB-Fim2 induced significant levels of Fim2-specific IgG in serum and bronchoalveolar lavage (BAL) and Fim2-specific IgA in BAL. Analysis of IgG isotypes and cytokines mRNA levels showed that CTB-Fim2 results in a mixed Th1/Th2 (T-helper) response. The data presented here provide support for CTB-Fim2 as a promising recombinant antigen against Bordetella pertussis infection. Noelia Olivera, Celina E. Castuma, Daniela Hozbor, María E. Gaillard, Martín Rumbo, and Ricardo M. Gómez Copyright © 2014 Noelia Olivera et al. All rights reserved. Naturally Occurring Compounds Elicit HIV-1 Replication in Chronically Infected Promonocytic Cells Mon, 12 May 2014 07:18:07 +0000 Since antiretroviral therapy suppresses but does not eradicate HIV-1 infection, methods to purge viral reservoirs are required. Many strategies involve the reactivation of chronically HIV infected cells to induce the expression of integrated viral genome. In this study, five bioactive compounds, the plant derivatives 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), nordihydroguaiaretic acid (NDGA), and curcumin (Cur) and the synthetic stigmasterol analogs (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) and (22S,23S)-3β-bromo-5α,22,23-trihydroxystigmastan-6-one (compound 2), were evaluated for their ability to elicit HIV replication in promonocytic (U1) and lymphocytic (H9+) HIV-1 chronically infected cells. The results revealed that natural compounds CDM, NDGA, and Cur were able to increase HIV-1 p24 antigen, determined by ELISA, only in latently infected promonocytic cells. CDM would reactivate HIV from latency by modulating the release of IL-6 and TNF-α, since the amount of both cytokines measured through ELISA significantly increased in U1 treated cells. Besides, NDGA increased ROS production, which might be related to the increase on p24 level observed in NDGA treated U1. These findings suggest that CDM, NDGA, and Cur might be candidates for further studies on latency-reversing therapeutics to eliminate latently HIV-1 reservoirs. Andrea Alejandra Barquero, María Eugenia Dávola, Diego Ariel Riva, Susana Esther Mersich, and Laura Edith Alché Copyright © 2014 Andrea Alejandra Barquero et al. All rights reserved. Atypical Enteropathogenic Escherichia coli Secretes Plasmid Encoded Toxin Sun, 11 May 2014 09:33:09 +0000 Plasmid encoded toxin (Pet) is a serine protease originally described in enteroaggregative Escherichia coli (EAEC) prototype strain 042 whose entire characterization was essentially obtained from studies performed with the purified toxin. Here we show that Pet is not exclusive to EAEC. Atypical enteropathogenic Escherichia coli (aEPEC) strains, isolated from diarrhea cases, express Pet and its detection in supernatants of infected HEp-2 cells coincides with the appearance of cell damage, which, in turn, were similar to those described with purified Pet. Pet secretion and the cytotoxic effects are time and culture medium dependent. In presence of DMEM supplemented with tryptone cell rounding and detachment were observed after just 5 h of incubation with the bacteria. In the absence of tryptone, the cytotoxic effects were detected only after 24 h of infection. We also show that, in addition to the prototype EAEC, other pet+ EAEC strains, also isolated from diarrhea cases, induce cellular damage in the same degree as the aEPEC. The cytotoxic effects of EAEC and aEPEC strains were significantly reduced in the presence of a serine protease inhibitor or anti-Pet IgG serum. Our results show a common aspect between the aEPEC and EAEC and provide the first evidence pointing to a role of Pet in aEPEC pathogenesis. Rita C. Ruiz, Keyde C. M. Melo, Sarita S. Rossato, Camila M. Barbosa, Lívia M. Corrêa, Waldir P. Elias, and Roxane M. F. Piazza Copyright © 2014 Rita C. Ruiz et al. All rights reserved. Characterization of the Opp Peptide Transporter of Corynebacterium pseudotuberculosis and Its Role in Virulence and Pathogenicity Thu, 08 May 2014 12:47:49 +0000 Despite the economic importance of caseous lymphadenitis (CLA), a chronic disease caused by Corynebacterium pseudotuberculosis, few genes related to the virulence of its etiologic agent have been characterized. The oligopeptide permease (Opp) transporters are located in the plasma membrane and have functions generally related to the uptake of peptides from the extracellular environment. These peptide transporters, in addition to having an important role in cell nutrition, also participate in the regulation of various processes involving intercellular signaling, including the control of the expression of virulence genes in pathogenic bacteria. To study the role of Opp in C. pseudotuberculosis, an OppD deficient strain was constructed via simple crossover with a nonreplicative plasmid carrying part of the oppD gene sequence. As occurred to the wild-type, the ΔoppD strain showed impaired growth when exposed to the toxic glutathione peptide (GSH), indicating two possible scenarios: (i) that this component can be internalized by the bacterium through an Opp-independent pathway or (ii) that there is toxicity while the peptide is extracellular. Additionally, the ΔoppD mutant presented a reduced ability to adhere to and infect macrophages compared to the wild-type, although both strains exhibit the same potential to colonize spleens and cause injury and death to infected mice. Pablo M. R. O. Moraes, Nubia Seyffert, Wanderson M. Silva, Thiago L. P. Castro, Renata F. Silva, Danielle D. Lima, Raphael Hirata Jr., Artur Silva, Anderson Miyoshi, and Vasco Azevedo Copyright © 2014 Pablo M. R. O. Moraes et al. All rights reserved. Xanthan Gum as an Adjuvant in a Subunit Vaccine Preparation against Leptospirosis Wed, 07 May 2014 09:09:57 +0000 Leptospiral immunoglobulin-like (Lig) proteins are of great interest due to their ability to act as mediators of pathogenesis, serodiagnostic antigens, and immunogens. Purified recombinant LigA protein is the most promising subunit vaccine candidate against leptospirosis reported to date, however, as purified proteins are weak immunogens the use of a potent adjuvant is essential for the success of LigA as a subunit vaccine. In the present study, we compared xanthan pv. pruni (strain 106), aluminium hydroxide (alhydrogel), and CpG ODN as adjuvants in a LigA subunit vaccine preparation. Xanthan gum is a high molecular weight extracellular polysaccharide produced by fermentation of Xanthomonas spp., a plant-pathogenic bacterium genus. Preparations containing xanthan induced a strong antibody response comparable to that observed when alhydrogel was used. Upon challenge with a virulent strain of L. interrogans serovar Copenhageni, significant protection (Fisher test, ) was observed in 100%, 100%, and 67% of hamsters immunized with rLigANI-xanthan, LigA-CpG-xanthan, and rLigANI-alhydrogel, respectively. Furthermore, xanthan did not cause cytotoxicity in Chinese hamster ovary (CHO) cells in vitro. The use of xanthan as an adjuvant is a novel alternative for enhancing the immunogenicity of vaccines against leptospirosis and possibly against other pathogens. Katia L. Bacelo, Daiane D. Hartwig, Fabiana K. Seixas, Rodrigo Schuch, Angelita da S. Moreira, Marta Amaral, Tiago Collares, Claire T. Vendrusculo, Alan J. A. McBride, and Odir A. Dellagostin Copyright © 2014 Katia L. Bacelo et al. All rights reserved. Bacterial Sepsis in Patients with Visceral Leishmaniasis in Northwest Ethiopia Tue, 06 May 2014 13:19:39 +0000 Background and Objectives. Visceral leishmaniasis (VL) is one of the neglected diseases affecting the poorest segment of world populations. Sepsis is one of the predictors for death of patients with VL. This study aimed to assess the prevalence and factors associated with bacterial sepsis, causative agents, and their antimicrobial susceptibility patterns among patients with VL. Methods. A cross-sectional study was conducted among parasitologically confirmed VL patients suspected of sepsis admitted to the University of Gondar Hospital, Northwest Ethiopia, from February 2012 to May 2012. Blood cultures and other clinical samples were collected and cultured following the standard procedures. Results. Among 83 sepsis suspected VL patients 16 (19.3%) had culture confirmed bacterial sepsis. The most frequently isolated organism was Staphylococcus aureus (68.8%; 11/16), including two methicillin-resistant isolates (MRSA). Patients with focal bacterial infection were more likely to have bacterial sepsis (). Conclusions. The prevalence of culture confirmed bacterial sepsis was high, predominantly due to S. aureus. Concurrent focal bacterial infection was associated with bacterial sepsis, suggesting that focal infections could serve as sources for bacterial sepsis among VL patients. Careful clinical evaluation for focal infections and prompt initiation of empiric antibiotic treatment appears warranted in VL patients. Mengistu Endris, Yegnasew Takele, Desalegn Woldeyohannes, Moges Tiruneh, Rezika Mohammed, Feleke Moges, Lutgarde Lynen, Jan Jacobs, Johan van Griensven, and Ermias Diro Copyright © 2014 Mengistu Endris et al. All rights reserved. Atypical Enteropathogenic Escherichia coli Strains form Biofilm on Abiotic Surfaces Regardless of Their Adherence Pattern on Cultured Epithelial Cells Tue, 06 May 2014 13:01:01 +0000 The aim of this study was to determine the capacity of biofilm formation of atypical enteropathogenic Escherichia coli (aEPEC) strains on abiotic and biotic surfaces. Ninety-one aEPEC strains, isolated from feces of children with diarrhea, were analyzed by the crystal violet (CV) assay on an abiotic surface after 24 h of incubation. aEPEC strains representing each HEp-2 cell type of adherence were analyzed after 24 h and 6, 12, and 18 days of incubation at 37°C on abiotic and cell surfaces by CFU/cm2 counting and confocal laser scanning microscopy (CLSM). Biofilm formation on abiotic surfaces occurred in 55 (60.4%) of the aEPEC strains. There was no significant difference in biofilm biomass formation on an abiotic versus prefixed cell surface. The biofilms could be visualized by CLSM at various developmental stages. aEPEC strains are able to form biofilm on an abiotic surface with no association with their adherence pattern on HEp-2 cells with the exception of the strains expressing UND (undetermined adherence). This study revealed the capacity of adhesion and biofilm formation by aEPEC strains on abiotic and biotic surfaces, possibly playing a role in pathogenesis, mainly in cases of persistent diarrhea. Hebert F. Culler, Cristiane M. Mota, Cecilia M. Abe, Waldir P. Elias, Marcelo P. Sircili, and Marcia R. Franzolin Copyright © 2014 Hebert F. Culler et al. All rights reserved. Effects of Shiga Toxin Type 2 on Maternal and Fetal Status in Rats in the Early Stage of Pregnancy Mon, 05 May 2014 14:32:30 +0000 Shiga toxin type 2 (Stx2), a toxin secreted by Shiga toxin-producing Escherichia coli (STEC), could be one of the causes of maternal and fetal morbimortality not yet investigated. In this study, we examined the effects of Stx2 in rats in the early stage of pregnancy. Sprague-Dawley pregnant rats were intraperitoneally (i.p.) injected with sublethal doses of Stx2, 0.25 and 0.5 ng Stx2/g of body weight (bwt), at day 8 of gestation (early postimplantation period of gestation). Maternal weight loss and food and water intake were analyzed after Stx2 injection. Another group of rats were euthanized and uteri were collected at different times to evaluate fetal status. Immunolocalization of Stx2 in uterus and maternal kidneys was analyzed by immunohistochemistry. The presence of Stx2 receptor (globotriaosylceramide, Gb3) in the uteroplacental unit was observed by thin layer chromatography (TLC). Sublethal doses of Stx2 in rats caused maternal weight loss and pregnancy loss. Stx2 and Gb3 receptor were localized in decidual tissues. Stx2 was also immunolocalized in renal tissues. Our results demonstrate that Stx2 leads to pregnancy loss and maternal morbidity in rats in the early stage of pregnancy. This study highlights the possibility of human pregnancy loss and maternal morbidity mediated by Stx2. Flavia Sacerdoti, María M. Amaral, Elsa Zotta, Ana M. Franchi, and Cristina Ibarra Copyright © 2014 Flavia Sacerdoti et al. All rights reserved. Classification of Epidemic Community-Acquired Methicillin-Resistant Staphylococcus aureus by Anatomical Site of Isolation Mon, 05 May 2014 14:16:36 +0000 Methicillin-resistant Staphylococcus aureus contributes significantly to cost, morbidity, and mortality due to infectious disease. We surveyed community-associated MRSA isolates to determine which strains were present within anatomical sites of interest. The most likely sources of MRSA among anatomic sites swabbed were wounds followed by the nasal cavity. The USA 300 MRSA strain was most commonly isolated among wound infections while nasal swabs largely yielded USA 100 MRSA. The frequency of isolation of USA 100 amongst community-associated strains is clinically significant as this strain is often correlated with invasive disease, exhibits broad antibiotic resistance, and has been considered to be hospital associated. The potential of USA 100 to cause serious disease and the frequency of its isolation suggest an important reservoir for opportunistic infection. These data demonstrate that MRSA epidemic clones are widespread among the community. Jill C. Roberts Copyright © 2014 Jill C. Roberts. All rights reserved. Identification of Virulence Factors Genes in Escherichia coli Isolates from Women with Urinary Tract Infection in Mexico Mon, 05 May 2014 13:43:46 +0000 E coli isolates (108) from Mexican women, clinically diagnosed with urinary tract infection, were screened to identify virulence genes, phylogenetic groups, and antibiotic resistance. Isolates were identified by MicroScan4 system; additionally, the minimum inhibitory concentration (MIC) was assessed. The phylogenetic groups and 16 virulence genes encoding adhesins, toxins, siderophores, lipopolysaccharide (LPS), and invasins were identified by PCR. Phylogenetic groups distribution was as follows: B1 9.3%, A 30.6%, B2 55.6%, and D 4.6%. Virulence genes prevalence was ecp 98.1%, fimH 86.1%, traT 77.8%, sfa/focDE 74.1%, papC 62%, iutA 48.1%, fyuA 44.4%, focG 2.8%, sfaS 1.9%, hlyA 7.4%, cnf-1 6.5%, cdt-B 0.9%, cvaC 2.8%, ibeA 2.8%, and rfc 0.9%. Regarding antimicrobial resistance it was above 50% to ampicillin/sulbactam, ampicillin, piperacillin, trimethoprim/sulfamethoxazole, ciprofloxacin, and levofloxacin. Uropathogenic E. coli clustered mainly in the pathogenic phylogenetic group B2. The isolates showed a high presence of siderophores and adhesion genes and a low presence of genes encoding toxins. The high frequency of papC gene suggests that these isolates have the ability to colonize the kidneys. High resistance to drugs considered as first choice treatment such as trimethoprim/sulfamethoxazole and fluoroquinolones was consistently observed. Daniela A. López-Banda, Erika M. Carrillo-Casas, Margarita Leyva-Leyva, Gabriel Orozco-Hoyuela, Ángel H. Manjarrez-Hernández, Sara Arroyo-Escalante, David Moncada-Barrón, Silvia Villanueva-Recillas, Juan Xicohtencatl-Cortes, and Rigoberto Hernández-Castro Copyright © 2014 Daniela A. López-Banda et al. All rights reserved. Phenotypic and Genotypic Characterization of Enterotoxigenic Escherichia coli Clinical Isolates from Northern Colombia, South America Wed, 30 Apr 2014 13:26:27 +0000 Enterotoxigenic Escherichia coli (ETEC) are major causes of childhood diarrhea in low and middle income countries including Colombia, South America. To understand the diversity of ETEC strains in the region, clinical isolates obtained from northern Colombia children were evaluated for multiple locus sequencing typing, serotyping, classical and nonclassical virulence genes, and antibiotic susceptibility. Among 40 ETEC clinical isolates evaluated, 21 (52.5%) were positive for LT gene, 13 (32.5%) for ST gene, and 6 (15%) for both ST and LT. The most prevalent colonization surface antigens (CS) were CS21 and CFA/I identified in 21 (50%) and 13 (32.5%) isolates, respectively. The eatA, irp2, and fyuA were the most common nonclassical virulence genes present in more than 60% of the isolates. Ampicillin resistance (80% of the strains) was the most frequent phenotype among ETEC strains followed by trimethoprim-sulfamethoxazole resistance (52.5%). Based on multiple locus sequencing typing (MLST), we recognize that 6 clonal groups of ETEC clinical isolates circulate in Colombia. ETEC clinical isolates from children in northern Colombia are highly diverse, yet some isolates circulating in the community belong to well-defined clonal groups that share a unique set of virulence factors, serotypes, and MLST sequence types. Julio A. Guerra, Yesenia C. Romero-Herazo, Octavio Arzuza, and Oscar G. Gómez-Duarte Copyright © 2014 Julio A. Guerra et al. All rights reserved. Falciparum Malaria as an Emerging Cause of Fever in Adults Living in Gabon, Central Africa Wed, 30 Apr 2014 06:36:25 +0000 Following the observed increase of malaria prevalence among older children in Gabon, a descriptive observational study was carried out in 2012 to determine the prevalence of malaria in adults presenting with fever in two health centres of Libreville, the capital city of Gabon. Thick- and thin-blood smears for malaria diagnosis were performed in febrile individuals aged more than 15 years old. Age, use of bed nets, previous antimalarial drug treatment, clinical symptoms, chest radiography results, and available haemoglobin data were also recorded. Among the 304 patients screened, the global malaria frequency was of 42.1% . Plasmodium (P). falciparum was the only species identified. The proportion of patients with a clinical malaria requiring parenteral treatment was 38.5%, whereas 47.5% of outpatients had uncomplicated malaria. According to WHO classification, 14 (19.7%) infected patients had severe malaria; neurological and respiratory symptoms tended to be more frequent in case of P. falciparum infection. Anaemia was found in 51.5% adults and none had severe anaemia. Almost half of adults consulting for fever in two health centres of the urban city of Libreville have malaria. The use of insecticide-treated bed nets, the screening, and the treatment of individuals with P. falciparum microscopic and submicroscopic asymptomatic infection or clinical malaria should be emphasized to reduce the transmission. Marielle K. Bouyou-Akotet, Christelle L. Offouga, Denise P. Mawili-Mboumba, Laurence Essola, Blondel Madoungou, and Maryvonne Kombila Copyright © 2014 Marielle K. Bouyou-Akotet et al. All rights reserved. Rural Residents in China Are at Increased Risk of Exposure to Tick-Borne Pathogens Anaplasma phagocytophilum and Ehrlichia chaffeensis Wed, 30 Apr 2014 00:00:00 +0000 As emerging tick born rickettsial diseases caused by A. phagocytophilum and E. chaffeensis, anaplasmosis and ehrlichiosis have become a serious threat to human and animal health throughout the world. In particular, in China, an unusual transmission of nosocomial cases of human granulocytic anaplasmosis occurred in Anhui Province in 2006 and more recent coinfection case of A. phagocytophilum and E. chaffeensis was documented in Shandong Province. Although the seroprevalence of human granulocytic anaplasmosis (former human granulocytic ehrlichiosis, HGE) has been documented in several studies, these data existed on local investigations, and also little data was reported on the seroprevalence of human monocytic ehrlichiosis (HME) in China. In this cross-sectional epidemiological study, indirect immunofluorescence antibody assay (IFA) proposed by WHO was used to detect A. phagocytophilum and E. chaffeensis IgG antibodies for 7,322 serum samples from agrarian residents from 9 provinces/cities and 819 urban residents from 2 provinces. Our data showed that farmers were at substantially increased risk of exposure. However, even among urban residents, risk was considerable. Seroprevalence of HGA and HME occurred in diverse regions of the country and tended to be the highest in young adults. Many species of ticks were confirmed carrying A. phagocytophilum organisms in China while several kinds of domestic animals including dog, goats, sheep, cattle, horse, wild rabbit, and some small wild rodents were proposed to be the reservoir hosts of A. phagocytophilum. The broad distribution of vector and hosts of the A. phagocytophilum and E. chaffeensis, especially the relationship between the generalized susceptibility of vectors and reservoirs and the severity of the disease’s clinical manifestations and the genetic variation of Chinese HGA isolates in China, is urgently needed to be further investigated. Lijuan Zhang, Hong Liu, Bianli Xu, Zhilun Zhang, Yuming Jin, Weiming Li, Qunying Lu, Liang Li, Litao Chang, Xiuchun Zhang, Desheng Fan, Minghua Cao, Manli Bao, Ying Zhang, Zengzhi Guan, Xueqin Cheng, Lina Tian, Shiwen Wang, Huilan Yu, Qiang Yu, Yong Wang, Yonggen Zhang, Xiaoyan Tang, Jieying Yin, Shijun Lao, Bin Wu, Juan Li, Weihong Li, Qiyi Xu, Yonglin Shi, and Fang Huang Copyright © 2014 Lijuan Zhang et al. All rights reserved. Virulence Factors Associated with Pediatric Shigellosis in Brazilian Amazon Tue, 29 Apr 2014 07:41:43 +0000 Shigellosis is a global human health problem and the incidence is highest among children. In the present work, main Shigella virulence genes was examined by PCR and compared to symptoms of pediatric shigellosis. Thirty Shigella isolates were identified from an etiologic study at which 1,339 children ranging 0–10 years old were enrolled. S. flexneri was the most frequent species reaching 60.0% of isolates, 22.2% were S. sonnei, and 6.6% were both S. dysenteriae and S. boydii. All Shigella infected children had diarrhea, but not all were accompanied by others symptoms of bacillary dysentery. Among major virulence genes, the PCR typing revealed ipaBCD was present in all isolates, followed by IpaH7.8, set-1A, set-1B, sen/ospD3, virF, and invE. The pathogenic potential of the ShET-1B subunit was observed in relation to dehydration () and ShET-2 related to the intestinal injury () evidenced by the presence of bloody diarrhea. Our results show associations among symptoms of shigellosis and virulence genes of clinical isolates of Shigella spp. Carolinie Batista Nobre da Cruz, Maria Carolina Scheffer de Souza, Paula Taquita Serra, Ivanildes Santos, Antonio Balieiro, Fabio Alessandro Pieri, Paulo Afonso Nogueira, and Patrícia Puccinelli Orlandi Copyright © 2014 Carolinie Batista Nobre da Cruz et al. All rights reserved. Analysis of the Virulence of an Atypical Enteropathogenic Escherichia coli Strain In Vitro and In Vivo and the Influence of Type Three Secretion System Mon, 28 Apr 2014 08:40:20 +0000 Atypical enteropathogenic Escherichia coli (aEPEC) inject various effectors into intestinal cells through a type three secretion system (T3SS), causing attaching and effacing (A/E) lesions. We investigated the role of T3SS in the ability of the aEPEC 1711-4 strain to interact with enterocytes in vitro (Caco-2 cells) and in vivo (rabbit ileal loops) and to translocate the rat intestinal mucosa in vivo. A T3SS isogenic mutant strain was constructed, which showed marked reduction in the ability to associate and invade but not to persist inside Caco-2 cells. After rabbit infection, only aEPEC 1711-4 was detected inside enterocytes at 8 and 24 hours pointing to a T3SS-dependent invasive potential in vivo. In contrast to aEPEC 1711-4, the T3SS-deficient strain no longer produced A/E lesions or induced macrophage infiltration. We also demonstrated that the ability of aEPEC 1711-4 to translocate through mesenteric lymph nodes to spleen and liver in a rat model depends on a functional T3SS, since a decreased number of T3SS mutant bacteria were recovered from extraintestinal sites. These findings indicate that the full virulence potential of aEPEC 1711-4 depends on a functional T3SS, which contributes to efficient adhesion/invasion in vitro and in vivo and to bacterial translocation to extraintestinal sites. Suely C. F. Sampaio, Fabiana C. Moreira, Ana M. A. Liberatore, Mônica A. M. Vieira, Terezinha Knobl, Fabiano T. Romão, Rodrigo T. Hernandes, Claudete S. A. Ferreira, Antônio P. Ferreira, Aloísio Felipe-Silva, Rita Sinigaglia-Coimbra, Ivan H. J. Koh, and Tania A. T. Gomes Copyright © 2014 Suely C. F. Sampaio et al. All rights reserved. Variations in KIR Genes: A Study in HIV-1 Serodiscordant Couples Sun, 27 Apr 2014 13:37:12 +0000 Background. NK cells have anti-HIV activity mediated through killer cell immunoglobulin-like receptors (KIRs). The current prospective cohort study evaluated whether variation in KIR genes is associated with HIV infection in discordant couples (DCs), where one spouse remains seronegative (HSN) despite repeated exposure to the HIV. Methods. KIR was genotyped using PCR SSP. Viral load and CD4 counts were estimated using commercially available reagents. Data were analyzed using SPSS software. Results. Among the 47 DCs, HSN spouses had significantly () higher frequencies of KIR3DS1. Regression analysis revealed significant () association of KIR2DS1 with low viral load. KIR2DS4 variant was associated () with high viral load. Three pairs of KIR genes were in strong LD in HSNs and two pairs in HSPs. There were 60 KIR genotypes, and 16 are reported the first time in the Indian population. Exclusive genotypes were present either in HSPs (, 11 unique genotypes) or in HSNs (, 9 unique genotypes). Conclusions. This study highlights for the first time in the Indian population an association of KIR genes in HIV infection where presence of exclusive and unique genotypes indicates possible association with either HIV infection or with protection. Vijay R. Chavan, Deepali Chaudhari, Swati Ahir, Zakiya Ansari, Preeti Mehta, and Jayanti Mania-Pramanik Copyright © 2014 Vijay R. Chavan et al. All rights reserved. Production and Characterization of a Polyclonal Antibody of Anti-rLipL21-IgG against Leptospira for Early Detection of Acute Leptospirosis Tue, 22 Apr 2014 08:50:04 +0000 Leptospirosis is one of the zoonotic diseases in animals and humans throughout the world. LipL21 is one of the important surface-exposed lipoproteins in leptospires and the most effective cross protective immunogenic antigen. It is widely considered as a diagnostic marker for leptospirosis. In this study, we evaluated the serodiagnostic potential of LipL21 protein of Leptospira interrogans serovar Pomona. We have successfully amplified, cloned, and expressed LipL21 in E. coli and evaluated its specificity by immunoblotting. Purified recombinant LipL21 (rLipL21) was inoculated into rabbits for the production of polyclonal antibody. Characterization of the purified IgG antibody against rLipL21 was performed by cross reactivity assay. Only sera from leptospirosis patients and rabbit hyperimmune sera recognized rLipL21 while the nonleptospirosis control sera showed no reaction in immunoblotting. We confirmed that anti-rLipL21-IgG antibody cross reacted with and detected only pathogenic leptospiral species and it did not react with nonpathogenic leptospires and other bacterial species. Results observed showed that anti-rLipL21-IgG antibody has high specificity and sensitivity to leptospires. The findings indicated that the antibody could be used in a diagnostic assay for detection of leptospires or their proteins in the early phase of infection. Arivudainambi Seenichamy, Abdul Rani Bahaman, Abdul Rahim Mutalib, and Siti Khairani-Bejo Copyright © 2014 Arivudainambi Seenichamy et al. All rights reserved. A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test Tue, 15 Apr 2014 14:05:52 +0000 A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible. Vanessa Suin, Florence Nazé, Aurélie Francart, Sophie Lamoral, Stéphane De Craeye, Michael Kalai, and Steven Van Gucht Copyright © 2014 Vanessa Suin et al. All rights reserved. Nonprofessional Phagocytic Cell Receptors Involved in Staphylococcus aureus Internalization Tue, 15 Apr 2014 09:56:18 +0000 Staphylococcus aureus is a successful human and animal pathogen. The majority of infections caused by this pathogen are life threatening, primarily because S. aureus has developed multiple evasion strategies, possesses intracellular persistence for long periods, and targets the skin and soft tissues. Therefore, it is very important to understand the mechanisms employed by S. aureus to colonize and proliferate in these cells. The aim of this review is to describe the recent discoveries concerning the host receptors of nonprofessional phagocytes involved in S. aureus internalization. Most of the knowledge related to the interaction of S. aureus with its host cells has been described in professional phagocytic cells such as macrophages. Here, we showed that in nonprofessional phagocytes the α5β1 integrin host receptor, chaperons, and the scavenger receptor CD36 are the main receptors employed during S. aureus internalization. The characterization and identification of new bacterial effectors and the host cell receptors involved will undoubtedly lead to new discoveries with beneficial purposes. Nayeli Alva-Murillo, Joel Edmundo López-Meza, and Alejandra Ochoa-Zarzosa Copyright © 2014 Nayeli Alva-Murillo et al. All rights reserved. Comparison of a New Gold Immunochromatographic Assay for the Rapid Diagnosis of the Novel Influenza A (H7N9) Virus with Cell Culture and a Real-Time Reverse-Transcription PCR Assay Mon, 14 Apr 2014 17:27:16 +0000 We assessed a colloidal gold immunochromatographic assay (GICA) for rapid detection of influenza A (H7N9) and compared it with reverse-transcription-polymerase chain reaction (RT-PCR) and viral culture. Samples from 35 H7N9 infected patients were collected, including 45 throat swab samples, 56 sputum samples, and 39 feces samples. All samples were tested by GICA, viral culture, and RT-PCR. GICA specifically reacted with recombinant HA proteins, virus lysates, and clinical samples from H7 subtype viruses. Compared with RT-PCR, GICA demonstrated low sensitivity (33.33%) but high specificity (97.56%). The positive rate of GICA tests for samples collected in the period from 8 to 21 days after contact with poultry was much higher than those for samples collected before or after this period. Compared with viral culture, GICA showed sensitivity of 91.67% and specificity of 82.03%. Sputum specimens were more likely to test positive for H7N9 virus than samples from throat swabs and feces. The GICA-based H7 test is a reliable, rapid, and convenient method for the screening and diagnosis of influenza A (H7N9) disease, especially for the sputum specimens with high viral load. It may be helpful in managing H7N9 epidemics and preliminary diagnosis in early stages in resource-limited settings. Changzhong Jin, Nanping Wu, Xiaorong Peng, Hangping Yao, Xiangyun Lu, Yu Chen, Haibo Wu, Tiansheng Xie, Linfang Cheng, Fumin Liu, Keren Kang, Shixing Tang, and Lanjuan Li Copyright © 2014 Changzhong Jin et al. All rights reserved. Development of a Colloidal Gold Kit for the Diagnosis of Severe Fever with Thrombocytopenia Syndrome Virus Infection Mon, 14 Apr 2014 09:08:17 +0000 It is critical to develop a cost-effective detection kit for rapid diagnosis and on-site detection of severe fever with thrombocytopenia syndrome virus (SFTSV) infection. Here, an immunochromatographic assay (ICA) to detect SFTSV infection is described. The ICA uses gold nanoparticles coated with recombinant SFTSV for the simultaneous detection of IgG and IgM antibodies to SFTSV. The ICA was developed and evaluated by using positive sera samples of SFTSV infection collected from the CDC of China. The reference laboratory diagnosis of SFTSV infection was based on the “gold standard”. The results demonstrated that the positive coincidence rate and negative coincidence rate were determined to be 98.4% and 100% for IgM and 96.7% and 98.6% for IgG, respectively. The kit showed good selectivity for detection of SFTSV-specific IgG and IgM with no interference from positive sera samples of Japanese encephalitis virus infection, Dengue virus infection, Hantavirus infection, HIV infection, HBV surface antigen, HCV antibody, Mycobacterium tuberculosis antibody, or RF. Based on these results, the ICS test developed may be a suitable tool for rapid on-site testing for SFTSV infections. Xianguo Wang, Quanfu Zhang, Fen Hao, Xunian Gao, Wei Wu, Minyao Liang, Zhihua Liao, Shuhong Luo, Weiwen Xu, Dexin Li, and Shiwen Wang Copyright © 2014 Xianguo Wang et al. All rights reserved. What Is New in Listeriosis? Mon, 14 Apr 2014 00:00:00 +0000 Listeriosis is a disease caused by Listeria monocytogenes (L. monocytogenes). L. monocytogenes is bacteria that usually infects some determined inhabitants, especially high risk patients such as the elderly, immunosuppressed patients and pregnant women. However, it can also affect people who do not have these risk factors. L. monocytogenes is widespread in nature being part of the faecal flora of many mammals and it is a common foodborne source. It is acquired by humans primarily through consumption of contaminated food. Besides, between 1% and 10% of the population is a faecal carrier of L.monocytogenes. Listeriosis may occur sporadically or in outbreaks. Infection causes a spectrum of illness, ranging from febrile gastroenteritis to invasive disease, including bacteraemia, sepsis, and meningoencephalitis. This infection has a low incidence, although it is undeniably increasing, particularly due to the rise of population of over 60 years old or of under 60 years olds with a predisposing condition. The diagnosis is complicated because of its incubation period and the different clinical manifestations. Also listeriosis has a high mortality despite adequate and early treatment. The importance of bacteraemia for L. monocytogenes lies in the infrequency of this bacterium and the high mortality, even with appropriate antibiotic treatment. Almudena Hernandez-Milian and Antoni Payeras-Cifre Copyright © 2014 Almudena Hernandez-Milian and Antoni Payeras-Cifre. All rights reserved. Human Antibody Response to Aedes albopictus Salivary Proteins: A Potential Biomarker to Evaluate the Efficacy of Vector Control in an Area of Chikungunya and Dengue Virus Transmission Sun, 13 Apr 2014 13:35:17 +0000 Aedes borne viruses represent public health problems in southern countries and threat to emerge in the developed world. Their control is currently based on vector population control. Much effort is being devoted to develop new tools to control such arbovirus. Recent findings suggest that the evaluation of human antibody (Ab) response to arthropod salivary proteins is relevant to measuring the level of human exposure to mosquito bites. Using an immunoepidemiological approach, the present study aimed to assess the usefulness of the salivary biomarker for measuring the efficacy of Ae. albopictus control strategies in La Reunion urban area. The antisaliva Ab response of adult humans exposed to Ae. albopictus was evaluatedbefore and after vector control measures. Our results showed a significant correlation between antisaliva Ab response and the level of exposure to vectors bites. The decrease of Ae. albopictus density has been detected by this biomarker two weeks after the implementation of control measures, suggesting its potential usefulness for evaluating control strategies in a short time period. The identification of species specific salivary proteins/peptides should improve the use of this biomarker. Souleymane Doucoure, François Mouchet, Sylvie Cornelie, Papa Makhtar Drame, Eric D’Ortenzio, Jean Sébastien DeHecq, and Franck Remoue Copyright © 2014 Souleymane Doucoure et al. All rights reserved. Insights on the Role of Antimicrobial Cuffed Endotracheal Tubes in Preventing Transtracheal Transmission of VAP Pathogens from an In Vitro Model of Microaspiration and Microbial Proliferation Thu, 10 Apr 2014 14:08:47 +0000 We developed an in vitro model to evaluate the effect of different cuffed endotracheal tubes (ETTs) on transtracheal transmission of ventilator-associated pneumonia (VAP) pathogens along external surfaces of ETTs. The model independently assessed the relative contributions of microbial proliferation to the distal tip and microaspiration of contaminated secretions past the cuff by testing in three modes: microaspiration only, microbial proliferation only, and simultaneous microaspiration and microbial proliferation. We evaluated transmission of methicillin resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa (PA) in the presence of a standard ETT; a soft, tapered cuff ETT with subglottic suctioning; and a novel antimicrobial gendine (combination of gentian violet and chlorhexidine) ETT in the model. In the microaspiration only mode, when leakage past the cuff occurred quickly, no ETT prevented transmission. When microaspiration was delayed, the gendine ETT was able to completely disinfect the fluid above the cuff and thereby prevent transmission of pathogens. In microbial proliferation only mode, the gendine ETT was the sole ETT that prevented transmission. With both mechanisms simultaneously available, transmission was dependent on how long microaspiration was delayed. Potent antimicrobial ETTs, such as a gendine ETT, can make unique contributions to prevent VAP when microaspiration is gradual. Joel Rosenblatt, Ruth Reitzel, Ying Jiang, Ray Hachem, and Issam Raad Copyright © 2014 Joel Rosenblatt et al. All rights reserved. Interactions of Neuropathogenic Escherichia coli K1 (RS218) and Its Derivatives Lacking Genomic Islands with Phagocytic Acanthamoeba castellanii and Nonphagocytic Brain Endothelial Cells Thu, 10 Apr 2014 10:04:34 +0000 Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α-hemolysin), adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (IbeA, CNF1), metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism) showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (CNF1), metabolism (D-serine catabolism) abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity. Farzana Abubakar Yousuf, Zuhair Yousuf, Junaid Iqbal, Ruqaiyyah Siddiqui, Hafsa Khan, and Naveed Ahmed Khan Copyright © 2014 Farzana Abubakar Yousuf et al. All rights reserved. Phylogenetic Analysis of Stenotrophomonas spp. Isolates Contributes to the Identification of Nosocomial and Community-Acquired Infections Thu, 10 Apr 2014 09:08:14 +0000 Stenotrophomonas ssp. has a wide environmental distribution and is also found as an opportunistic pathogen, causing nosocomial or community-acquired infections. One species, S. maltophilia, presents multidrug resistance and has been associated with serious infections in pediatric and immunocompromised patients. Therefore, it is relevant to conduct resistance profile and phylogenetic studies in clinical isolates for identifying infection origins and isolates with augmented pathogenic potential. Here, multilocus sequence typing was performed for phylogenetic analysis of nosocomial isolates of Stenotrophomonas spp. and, environmental and clinical strains of S. maltophilia. Biochemical and multidrug resistance profiles of nosocomial and clinical strains were determined. The inferred phylogenetic profile showed high clonal variability, what correlates with the adaptability process of Stenotrophomonas to different habitats. Two clinical isolates subgroups of S. maltophilia sharing high phylogenetic homogeneity presented intergroup recombination, thus indicating the high permittivity to horizontal gene transfer, a mechanism involved in the acquisition of antibiotic resistance and expression of virulence factors. For most of the clinical strains, phylogenetic inference was made using only partial ppsA gene sequence. Therefore, the sequencing of just one specific fragment of this gene would allow, in many cases, determining whether the infection with S. maltophilia was nosocomial or community-acquired. Vinicius Godoy Cerezer, Silvia Yumi Bando, Jacyr Pasternak, Marcia Regina Franzolin, and Carlos Alberto Moreira-Filho Copyright © 2014 Vinicius Godoy Cerezer et al. All rights reserved. PCR and Microscopic Identification of Isolated Leishmania tropica from Clinical Samples of Cutaneous Leishmaniasis in Human Population of Kohat Region in Khyber Pakhtunkhwa Tue, 01 Apr 2014 16:21:24 +0000 Leishmania tropica was isolated from the clinical patients of cutaneous leishmaniasis in rural community of Kohat district in Khyber Pakhtunkhwa province and was identified through PCR, microscopy, and culture techniques. A total of 113 samples from the clinical patients were examined through PCR, microscopy, and culture which showed 87.61% (99/113), 53.98% (61/113), and 46.90% (53/113) prevalence. During the study, 186 bp Leishmania tropica was identified through PCR. Thus the sensitivity of PCR is very high as compared to the conventional techniques. Nasser M. Abd El-Salam, Sultan Ayaz, and Riaz Ullah Copyright © 2014 Nasser M. Abd El-Salam et al. All rights reserved. The Function of CD3+CD56+ NKT-Like Cells in HIV-Infected Individuals Thu, 20 Mar 2014 08:42:03 +0000 CD3+CD56+ NKT-like cells are one of the critical effectors in the immune response to viral infection and tumors, but the functional features of NKT-like cells in HIV infection have been rarely reported. In this study, we observed and described the state of NKT-like cell functions in primary HIV-infected individuals (PHIs), chronic HIV-infected individuals (CHIs), long-term nonprogressors (LTNPs), and HIV-negative controls (NCs). The results showed that the percentage of IFN-γ+CD3+CD56+ NKT-like cells was notably higher in LTNPs compared with CHIs, and the proportion of CD3+CD56+ NKT-like cells with dual function (IFN-γ+CD107a+ NKT-like cells) in LTNPs was also much higher than in CHIs. Additionally, the percentages of IFN-γ+CD107a+ NKT-like cells negatively correlated with viral load. Taken together, our data demonstrated that good functions of CD3+CD56+ NKT-like cells in LTNPs likely occurred as a protective mechanism that slows down HIV disease progression. Yongjun Jiang, Xiaojian Cui, Chen Cui, Jian Zhang, Fangyuan Zhou, Zining Zhang, Yajing Fu, Junjie Xu, Zhenxing Chu, Jing Liu, Xiaoxu Han, Christina Liao, Yanan Wang, Yaming Cao, and Hong Shang Copyright © 2014 Yongjun Jiang et al. All rights reserved. TB Meningitis in HIV-Positive Patients in Europe and Argentina: Clinical Outcome and Factors Associated with Mortality Tue, 31 Dec 2013 10:18:23 +0000 Objectives. The study aimed at describing characteristics and outcome of tuberculous meningitis (TBM) in HIV-positive patients and comparing these parameters with those of extrapulmonary TB (TBEP) and pulmonary TB (TBP). Methods. Kaplan-Meier estimation and Poisson regression models were used to assess the mortality following TB diagnosis and to evaluate potential prognostic factors for the 3 groups of TB patients separately. Results. A total of 100 patients with TBM, 601 with TBEP, and 371 TBP were included. Patients with TBM had lower CD4 cell counts and only 17.0% received antiretroviral therapy (ART) at TB diagnosis. The cumulative probability of death at 12 months following TB was 51.2% for TBM (95% CI 41.4–61.6%), 12.3% for TBP (8.9–15.7%), and 19.4% for TBEP (16.1–22.6) (; log-rank test). For TBM, factors associated with a poorer prognosis were not being on ART (adjusted incidence rate ratio (aIRR) 4.00 (1.72–9.09), a prior AIDS diagnosis ( (2.61–8.92)), and receiving care in Eastern Europe ( (2.58–11.34))). Conclusions. TBM among HIV-positive patients was associated with a high mortality rate, especially for patients from Eastern Europe and patients with advanced HIV-infection, which urgently calls for public health interventions to improve both TB and HIV aspects of patient management. Anne Marie W. Efsen, Alexander M. Panteleev, Daniel Grint, Daria N. Podlekareva, Anna Vassilenko, Aza Rakhmanova, Indra Zeltina, Marcelo H. Losso, Robert F. Miller, Enrico Girardi, Joan Caylá, Frank A. Post, Jose M. Miro, Mathias Bruyand, Hansjakob Furrer, Niels Obel, Jens D. Lundgren, Amanda Mocroft, Ole Kirk, and HIV/TB Study Group Copyright © 2013 Anne Marie W. Efsen et al. All rights reserved. Pediatric Tuberculosis in Young Children in India: A Prospective Study Tue, 10 Dec 2013 09:28:33 +0000 Background. India has one of the highest tuberculosis (TB) burdens globally. However, few studies have focused on TB in young children, a vulnerable population, where lack of early diagnosis results in poor outcomes. Methods. Young children (≤5 years) with suspected TB were prospectively enrolled at a tertiary hospital in Pune, India. Detailed clinical evaluation, HIV testing, mycobacterial cultures, and drug susceptibility testing were performed. Results. 223 children with suspected TB were enrolled. The median age was 31 months, 46% were female, 86% had received BCG, 57% were malnourished, and 10% were HIV positive. 12% had TB disease (definite or probable), 35% did not have TB, while TB could not be ruled out in 53%. Extrapulmonary disease was noted in 46%, which was predominantly meningeal. Tuberculin skin test (TST) was positive in 20% of children with TB. Four of 7 (57%) children with culture-confirmed TB harbored drug-resistant (DR) strains of whom 2 (50%) were multi-DR (MDR). In adjusted analyses, HIV infection, positive TST, and exposure to household smoke were found to be significantly associated with children with TB (). Mortality (at 1 year) was 3 of 26 (12%) and 1 of 79 (1%), respectively, in children with TB and those without TB (). Conclusions. Diagnosis of TB is challenging in young children, with high rates of extra-pulmonary and meningeal disease. While the data on DR-TB are limited by the small sample size, they are however concerning, and additional studies are needed to more accurately define the prevalence of DR strains in this vulnerable population. Sanjay K. Jain, Alvaro Ordonez, Aarti Kinikar, Nikhil Gupte, Madhuri Thakar, Vidya Mave, Jennifer Jubulis, Sujata Dharmshale, Shailaja Desai, Swarupa Hatolkar, Anju Kagal, Ajit Lalvani, Amita Gupta, and Renu Bharadwaj Copyright © 2013 Sanjay K. Jain et al. All rights reserved. Circulation of HIV-1 CRF02_AG among MSM Population in Central Italy: A Molecular Epidemiology-Based Study Thu, 28 Nov 2013 17:59:02 +0000 Introduction. The evolutionary and demographic history of the circular recombinant form CRF02_AG in a selected retrospective group of HIV-1 infected men who have sex with men (MSM) resident in Central Italy was investigated. Methods. A total of 55 HIV-1 subtype CRF02_AG pol sequences were analyzed using Bayesian methods and a relaxed molecular clock to reconstruct their dated phylogeny and estimate population dynamics. Results. Dated phylogeny indicated that the HIV-1 CRF02_AG strains currently circulating in Central Italy originated in the early 90's. Bayesian phylogenetic analysis revealed the existence of a main HIV-1 CRF02_AG clade, introduced in the area of Rome before 2000 and subsequently differentiated in two different subclades with a different date of introduction (2000 versus 2005). All the sequences within clusters were interspersed, indicating that the MSM analyzed form a close and restricted network where the individuals, also moving within different clinical centers, attend the same places to meet and exchange sex. Conclusions. It was suggested that the HIV-1 CRF02_AG epidemic entered central Italy in the early 1990s, with a similar trend observed in western Europe. Massimo Giuliani, Maria M. Santoro, Alessandra Lo Presti, Eleonora Cella, Paola Scognamiglio, Alessia Lai, Alessandra Latini, Lavinia Fabeni, Caterina Gori, Carmela Pinnetti, Enrico Girardi, Carlo F. Perno, Gianguglielmo Zehender, and Massimo Ciccozzi Copyright © 2013 Massimo Giuliani et al. All rights reserved. Pattern Recognition Receptors and Cytokines in Mycobacterium tuberculosis Infection—The Double-Edged Sword? Tue, 12 Nov 2013 13:40:38 +0000 Tuberculosis, an infectious disease caused by Mycobacterium tuberculosis (Mtb), remains a major cause of human death worldwide. Innate immunity provides host defense against Mtb. Phagocytosis, characterized by recognition of Mtb by macrophages and dendritic cells (DCs), is the first step of the innate immune defense mechanism. The recognition of Mtb is mediated by pattern recognition receptors (PRRs), expressed on innate immune cells, including toll-like receptors (TLRs), complement receptors, nucleotide oligomerization domain like receptors, dendritic cell-specific intercellular adhesion molecule grabbing nonintegrin (DC-SIGN), mannose receptors, CD14 receptors, scavenger receptors, and FC receptors. Interaction of mycobacterial ligands with PRRs leads macrophages and DCs to secrete selected cytokines, which in turn induce interferon-- (IFN-) dominated immunity. IFN and other cytokines like tumor necrosis factor- (TNF) regulate mycobacterial growth, granuloma formation, and initiation of the adaptive immune response to Mtb and finally provide protection to the host. However, Mtb can evade destruction by antimicrobial defense mechanisms of the innate immune system as some components of the system may promote survival of the bacteria in these cells and facilitate pathogenesis. Thus, although innate immunity components generally play a protective role against Mtb, they may also facilitate Mtb survival. The involvement of selected PRRs and cytokines on these seemingly contradictory roles is discussed. Md. Murad Hossain and Mohd-Nor Norazmi Copyright © 2013 Md. Murad Hossain and Mohd-Nor Norazmi. All rights reserved. Predictors of Delayed Antiretroviral Therapy Initiation, Mortality, and Loss to Followup in HIV Infected Patients Eligible for HIV Treatment: Data from an HIV Cohort Study in India Tue, 29 Oct 2013 15:21:34 +0000 Studies from Sub-Saharan Africa have shown that a substantial number of HIV patients eligible for antiretroviral therapy (ART) do not start treatment. However, data from other low- or middle-income countries are scarce. In this study, we describe the outcomes of 4105 HIV patients who became ART eligible from January 2007 to November 2011 in an HIV cohort study in India. After three years of ART eligibility, 78.4% started ART, 9.3% died before ART initiation, and 10.3% were lost to followup. Diagnosis of tuberculosis, being homeless, lower CD4 count, longer duration of pre-ART care, belonging to a disadvantaged community, being widowed, and not living near a town were associated with delayed ART initiation. Diagnosis of tuberculosis, being homeless, lower CD4 count, shorter duration of pre-ART care, belonging to a disadvantaged community, illiteracy, and age >45 years were associated with mortality. Being homeless, being single, not living near a town, having a CD4 count <150 cells/μL, and shorter duration of pre-ART care were associated with loss to followup. These results highlight the need to improve the timely initiation of ART in HIV programmes in India, especially in ART eligible patients with tuberculosis, low CD4 counts, living in rural areas, or having a low socioeconomic status. Gerardo Alvarez-Uria, Raghavakalyan Pakam, Manoranjan Midde, and Praveen Kumar Naik Copyright © 2013 Gerardo Alvarez-Uria et al. All rights reserved. Evaluation of hsp65 Nested PCR-Restriction Analysis (PRA) for Diagnosing Tuberculosis in a High Burden Country Thu, 24 Oct 2013 10:55:18 +0000 Current study evaluated the hsp65 Nested PCR Restriction Fragment Length Polymorphism Analysis (hsp65 Nested PCR-PRA) to detect and identify Mycobacterium tuberculosis complex directly in clinical samples for a rapid and specific diagnosis of tuberculosis (TB). hsp65 Nested PCR-PRA was applied directly to 218 clinical samples obtained from 127 patients suspected of TB or another mycobacterial infection from July 2009 to July 2010. The hsp65 Nested PCR-PRA showed 100% sensitivity and 95.0 and 93.1% specificity in comparison with culture and microscopy (acid fast bacillus smear), respectively. hsp65 Nested PCR-PRA was shown to be a fast and reliable assay for diagnosing TB, which may contribute towards a fast diagnosis that could help the selection of appropriate chemotherapeutic and early epidemiological management of the cases which are of paramount importance in a high TB burden country. Sara Macente, Clarice Queico Fujimura Leite, Adolfo Carlos Barreto Santos, Vera Lúcia Dias Siqueira, Luzia Neri Cosmo Machado, Nadir Rodrigues Marcondes, Mario Hiroyuki Hirata, Rosário Dominguez Crespo Hirata, and Rosilene Fressatti Cardoso Copyright © 2013 Sara Macente et al. All rights reserved. Frequency and Characteristics of Infections Caused by Extended-Spectrum Beta-Lactamase-Producing Organisms in Neonates: A Prospective Cohort Study Tue, 24 Sep 2013 15:25:39 +0000 This prospective cohort study was conducted to determine the frequency of infections caused by extended-spectrum beta-lactamase- (ESBL-) producing organisms, various bacteria producing ESBL, antibiotic susceptibility of these organisms, and the risk factors associated with these infections in a neonatal intensive care unit in a tertiary care hospital in North India. Of the 150 neonates enrolled in the study, 47 culture-positive neonates were included in the study cohort and were divided into two groups: ESBL-positive (8 neonates) and ESBL-negative (39 neonates) cohorts. Various organisms were isolated from 72 culture samples in these 47 neonates. Of these, 10 culture samples grew ESBL-positive organisms and 62 samples grew ESBL-negative organisms. The frequency of ESBL-producing organisms was found to be 5.3%. ESBL infection incidence densities were found to be 3.4 per 1000 patient-days. Klebsiella (60%) was the most common organism producing ESBL followed by Escherichia coli (30%) and Pseudomonas (10%). Eighty percent of the ESBL-producing organisms were sensitive to piperacillin-tazobactam. Risk factors found significant by univariate analysis () were preterm, low birthweight, perinatal asphyxia, respiratory distress syndrome, anaemia, metabolic acidosis, prolonged mechanical ventilation (>7 days), length of hospitalization, length of level 3 stay, prior antibiotic use, central venous catheter duration, peripherally inserted central venous catheter duration, and total parenteral nutrition duration. Factors that retained significance in the logistic regression model were duration of hospital stay (adjusted OR: 0.958, CI: 0.920–0.997, and value = 0.037) and gestational age (adjusted OR: 1.39, CI: 1.037–1.865, and value = 0.028). There was no significant difference in the mortality between the two groups. Nandini Vijayakanthi, Dheeraj Bahl, Nirmaljit Kaur, Arti Maria, and Nand Kishore Dubey Copyright © 2013 Nandini Vijayakanthi et al. All rights reserved. Hyaluronic Acid Derived from Other Streptococci Supports Streptococcus pneumoniae In Vitro Biofilm Formation Thu, 19 Sep 2013 08:24:42 +0000 We investigate the role of hyaluronic acid (HA) on S. pneumoniae in vitro biofilm formation and evaluate gene expressions of virulence and/or biofilm related genes. Biofilms were grown in medium supplied with HA derived from capsule of Streptococcus equi. The biomasses of biofilms were detected by crystal-violet (CV) microtiter plate assay, and the morphology was viewed under scanning electron microscope (SEM). The gene expressions were assessed by relative quantitative RT-PCR. The results showed that the HA support pneumococcal growth in planktonic form and within biofilms. The CV-microtiter plate assay detected significantly increased biofilm growth in medium containing HA. The SEM analysis revealed thick and organized biofilms in positive control and HA supplemented medium. The nanA, nanB, bgaA, strH, luxS, hysA, ugl, and PST-EIIA encoding genes were significantly upregulated in the planktonic cells grown in presence of HA, while the lytA and comA genes were downregulated. Similarly the luxS, hysA, ugl, and PST-EIIA encoding genes were significantly upregulated by more than 2-folds in HA biofilms. The results of this study indicate that the HA derived from capsule of S. equi supports pneumococcal growth in planktonic state and within biofilms and upregulated virulence and biofilm related genes. Mukesh Kumar Yadav, Sung-Won Chae, Kyeongsoon Park, and Jae-Jun Song Copyright © 2013 Mukesh Kumar Yadav et al. All rights reserved. Listeria monocytogenes Meningitis in Adults: The Czech Republic Experience Wed, 11 Sep 2013 14:05:19 +0000 Background. Listeria monocytogenes (LM) is currently the third most frequent pathogen of bacterial meningitis in adults. Methods. A prospective study of patients with LM meningitis in a Czech tertiary care hospital, carried out from 1997 to 2012. Results. Thirty-one patients were diagnosed with LM meningitis, which was 7% of a total of 440 adult patients with acute bacterial meningitis (ABM) over a 16-year period. Their median age was 63 years, range 26–80 years. Nineteen patients (61%) had underlying immunocompromising comorbidity; 15 patients (48%) were older than 65 years. Fourteen patients (45%) had arterial hypertension. The typical triad of fever, neck stiffness, and altered mental status was present in 21 patients (68%). The median count of cerebrospinal fluid (CSF) leukocytes was 680/L, protein level 2.6 g/L, and glucose ratio 0.28. Four patients (13%) died, and nine (29%) survived with moderate to severe sequelae. Conclusion. LM meningitis is known to affect immunosuppressed and elderly patients. Arterial hypertension seems to be another important predisposing factor. Clinical symptoms, CSF findings, and disease outcomes, did not significantly differ from other community-acquired ABM in our study, although the CSF leukocyte count was lower. Ampicillin showed good clinical and bacteriological efficacy in the majority of patients. Olga Dzupova, Hanus Rozsypal, Dita Smiskova, and Jiri Benes Copyright © 2013 Olga Dzupova et al. All rights reserved. Emergence in Western African Countries of MDR-TB, Focus on Côte d’Ivoire Wed, 04 Sep 2013 13:43:45 +0000 Tuberculosis (TB) is responsible for a high mortality rate (2.5%) worldwide, mainly in developing countries with a high prevalence of human immunodeficiency virus (HIV). The emergence of multiresistant strains of TB poses an extreme risk for TB outbreaks and highlights the need for global TB control strategies. Among Western African countries, Côte d’Ivoire (CI) represents a specific example of a country with great potential to prevent TB. Specifically, CI has a promising healthcare system for monitoring diseases, including vaccination programs. However, military and political conflict in CI favors the spread of infectious diseases, TB being among the most devastating. Compilation of the studies identifying common causes of TB would be extremely beneficial for the development of treatment and prevention strategies. Therefore, the purpose of this comprehensive review is to evaluate the epidemiology of TB in CI, describe the factors involved in pathogenesis, and suggest simple and applicable prevention strategies. Euloge Ekaza, Raymond Kouassi N’Guessan, Adèle Kacou-N’Douba, N’Guetta Aka, Jacquemin Kouakou, Françoise Le Vacon, Fréderic Altare, Gilles Potel, and Marie-France de-La-Cochetiere Copyright © 2013 Euloge Ekaza et al. All rights reserved. Predictors of Attrition in Patients Ineligible for Antiretroviral Therapy after Being Diagnosed with HIV: Data from an HIV Cohort Study in India Thu, 29 Aug 2013 14:50:25 +0000 In newly HIV-diagnosed patients, the CD4+ lymphocyte count is measured to determine the need for antiretroviral therapy (ART). Studies from Sub-Saharan Africa have shown that patients who are ART ineligible at the first assessment have poor retention in care, but data from other low- or middle-income countries are scarce. In this study we describe the retention in pre-ART care of 1696 patients who were ineligible for ART after being diagnosed with HIV in a cohort study in India. More than one-third of ART ineligible patients had poor retention in care, and the attrition was higher in those with longer follow-up periods. Of those patients with poor retention, only 10% came back to the clinics, and their CD4 cell counts were lower than the ones of patients retained in care. After 4.5 years of follow-up, the cumulative incidence of loss to follow-up was 50%. Factors associated with attrition were being homeless, being illiterate, belonging to a disadvantaged community, being symptomatic at the time of the HIV diagnosis, male gender, and not living near a town. Widows were given nutritional support and, therefore, had better retention in care. The results of this study highlight the need to improve the retention in care of ART ineligible patients in India. Gerardo Alvarez-Uria, Manoranjan Midde, Raghavakalyan Pakam, and Praveen Kumar Naik Copyright © 2013 Gerardo Alvarez-Uria et al. All rights reserved. A Qualitative Study of Barriers to Enrollment into Free HIV Care: Perspectives of Never-in-Care HIV-Positive Patients and Providers in Rakai, Uganda Thu, 22 Aug 2013 10:26:08 +0000 Background. Early entry into HIV care is low in Sub-Saharan Africa. In Rakai, about a third (31.5%) of HIV-positive clients who knew their serostatus did not enroll into free care services. This qualitative study explored barriers to entry into care from HIV-positive clients who had never enrolled in care and HIV care providers. Methods. We conducted 48 in-depth interviews among HIV-infected individuals aged 15–49 years, who had not entered care within six months of result receipt and referral for free care. Key-informant interviews were conducted with 12 providers. Interviews were audio-recorded and transcripts subjected to thematic content analysis based on the health belief model. Results. Barriers to using HIV care included fear of stigma and HIV disclosure, women’s lack of support from male partners, demanding work schedules, and high transport costs. Programmatic barriers included fear of antiretroviral drug side effects, long waiting and travel times, and inadequate staff respect for patients. Denial of HIV status, belief in spiritual healing, and absence of AIDS symptoms were also barriers. Conclusion. Targeted interventions to combat stigma, strengthen couple counseling and health education programs, address gender inequalities, and implement patient-friendly and flexible clinic service hours are needed to address barriers to HIV care. Gertrude Nakigozi, Lynn Atuyambe, Moses Kamya, Fredrick E. Makumbi, Larry W. Chang, Neema Nakyanjo, Godfrey Kigozi, Fred Nalugoda, Valerian Kiggundu, David Serwadda, Maria Wawer, and Ronald Gray Copyright © 2013 Gertrude Nakigozi et al. All rights reserved. Mediterranean Spotted Fever in Southeastern Romania Sun, 18 Aug 2013 08:52:32 +0000 Although cases of Mediterranean spotted fever (MSF) have been reported for decades in southeastern Romania, there are few published data. We retrospectively studied 339 patients, diagnosed with MSF at the National Institute of Infectious Diseases “Prof. Dr. Matei Bals” between 2000 and 2011, in order to raise awareness about MSF in certain regions of Romania. According to the Raoult diagnostic criteria 171 (50.4%) had a score >25 points. Mean age was 52.5 years. One hundred and fifty-five (90.6%) patients were from Bucharest and the surrounding region. Almost all patients presented with fever (99.4%) and rash (98.2%), and 57.9% had evidence of a tick bite. There were no recorded deaths. Serologic diagnosis was made by indirect immunofluorescence assay. Of the 171 patients, serology results for R. conorii were available in 147. One hundred and twenty-three (83.7%) of them had a titer IgG ≥1 : 160 or a fourfold increase in titer in paired samples. MSF is endemic in southeastern Romania and should be considered in patients with fever and rash even in the absence of recognized tick exposure. Since the disease is prevalent in areas highly frequented by tourists, travel-associated MSF should be suspected in patients with characteristic symptoms returning from the endemic area. Daniela Pitigoi, Ioana D. Olaru, Daniela Badescu, Alexandru Rafila, Victoria Arama, and Adriana Hristea Copyright © 2013 Daniela Pitigoi et al. All rights reserved. HBV Induced HCC: Major Risk Factors from Genetic to Molecular Level Wed, 07 Aug 2013 15:54:08 +0000 Hepatocellular carcinoma (HCC) is a deadly and emerging disease leading to death in Asian countries. High hepatitis B virus (HBV) load and chronic hepatitis B (CHB) infection increase the risk of developing HCC. HBV is a DNA virus that can integrate DNA into host genome thereby increase the yield of transactivator protein HBxAg that may deregulate many pathways involving in metabolism of cells. Several monogenic and polygenic risk factors are also involved in HCC development. This review summarizes the mechanism involved in HCC development and discusses some promising therapies to make HCC curative. Ambreen Ayub, Usman Ali Ashfaq, and Asma Haque Copyright © 2013 Ambreen Ayub et al. All rights reserved. A Two-Tube Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Sixteen Human Respiratory Virus Types/Subtypes Mon, 05 Aug 2013 09:44:42 +0000 There is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens. With an intended application in provincial Centers for Diseases Control and Prevention, in this study, we present a two-tube multiplex RT-PCR assay (two-tube assay) using automatic electrophoresis to simultaneously detect sixteen common respiratory viruses. The specificity and the sensitivity of the assay were tested. The assay could detect 20–200 copies per reaction when each viral type was assayed individually, 2000 copies with 9 premixed viral targets in the multiplexed assay in tube 1, and 200 copies with 8 premixed templates in tube 2. A total of 247 specimens were used to evaluate the two-tube assay, and the results were compared with those obtained from the Luminex xTAG RVP Fast assay. The discordant results were confirmed by sequencing or by the Seeplex RV15 ACE detection kit. There were no false positives, but six false negatives occurred with the two-tube assay. In conclusion, the two-tube assay is demonstrated to have great potential for routine surveillance of respiratory virus infection in China. Jin Li, Shunxiang Qi, Chen Zhang, Xiumei Hu, Hongwei Shen, Mengjie Yang, Ji Wang, Miao Wang, Wenbo Xu, and Xuejun Ma Copyright © 2013 Jin Li et al. All rights reserved. Secondary Bacterial Infections in Patients with Seasonal Influenza A and Pandemic H1N1 Thu, 20 Jun 2013 13:17:01 +0000 The aim of the present study is to analyse the secondary bacterial infections in a large group of patients with seasonal influenza A and influenza A(H1N1) pdm09. Patients diagnosed with seasonal influenza A and influenza A(H1N1) pdm09 between 2005 and 2009 were enrolled in the study. Data was retrieved from medical records and laboratory information systems (LIS). In total, 1094 patients with laboratory confirmed influenza were studied. There were 352 patients with seasonal influenza A and 742 patients with influenza A(H1N1) pdm09. The patients with influenza A were older and had higher comorbidity than patients with influenza A(H1N1) pdm09 ( and , resp.). Hospital admission was higher in influenza A group (). In contrast, ICU admission was higher in patients with influenza A(H1N1) pdm09 than influenza A patients (). There were higher numbers of bacterial samples taken and culture positivity in patients with influenza A than patients with influenza A(H1N1) pdm09 ( and , resp.). In both groups, the majority of the patients with positive bacterial cultures had underlying diseases. The present study shows that the patient characteristics and the frequency of secondary bacterial infections were different in patients with seasonal influenza A and in patients with influenza A(H1N1) pdm09. Karin Liderot, Marcus Ahl, and Volkan Özenci Copyright © 2013 Karin Liderot et al. All rights reserved. Mucosal Leishmaniasis: An Underestimated Presentation of a Neglected Disease Tue, 18 Jun 2013 16:36:22 +0000 We present a review of current knowledge about mucosal leishmaniasis (ML). Although involvement of mucous membranes is classically admitted in New World leishmaniasis, particularly occurring in infection by Leishmania (L.) braziliensis species complex, ML is also a possible presentation of Old World leishmaniasis, in either L. donovani or L. major species complex infections. Thus, ML has to be considered not only as a Latin American disease but as an Old and New World disease. We describe ML epidemiology, pathogenesis, clinics, diagnosis, and therapy. Considering both its highly disfiguring lesions and its possible lethal outcome, ML should not be underestimated by physicians. Moreover, leishmaniasis is expected to increase its burden in many countries as sandfly vector distribution is widespreading towards non-endemic areas. Finally, the lack of clear understanding of ML pathogenesis and the absence of effective human vaccines strongly claim for more research. Alessio Strazzulla, Salvatore Cocuzza, Marilia Rita Pinzone, Maria Concetta Postorino, Stefano Cosentino, Agostino Serra, Bruno Cacopardo, and Giuseppe Nunnari Copyright © 2013 Alessio Strazzulla et al. All rights reserved. NRAMP1, VDR, HLA-DRB1, and HLA-DQB1 Gene Polymorphisms in Susceptibility to Tuberculosis among the Chinese Kazakh Population: A Case-Control Study Mon, 10 Jun 2013 10:01:54 +0000 Background. To explore the potential role of natural-resistance-associated macrophage protein 1 (NRAMP1) gene, vitamin D receptor (VDR) gene, (human leukocyte antigen, (HLA-DRB1) HLA) -DRB1 gene, and HLA-DQB1 gene polymorphisms in susceptibility to tuberculosis (TB) in the Chinese Kazakh population. Methods. A case-control study was performed on the Chinese Kazak population. Genetic polymorphisms of NRAMP1 gene (3′UTR) and VDR gene (TaqI and FokI) were analysed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing analysis in TB patients and healthy controls. Genetic polymorphisms of HLA-DRB1 gene and HLA-DQB1 gene in the two groups were detected with polymerase chain reaction-sequence-specific primers (PCR-SSPs) technique and sequencing analysis. Results. There was statistically significant difference in the 3′UTR polymorphism between the TB patients and healthy controls in the Chinese Kazak population (; OR = 1.859; 95% CI = 1.182–2.926). Significant difference was observed in the FokI polymorphism between the TB patients and healthy controls (; OR = 1.530; 95% CI = 1.007–2.325). It does not disclose any significant association between the disease and TaqI (). Alleles HLA-DRB1*04 and HLA-DQB1*0201 occurred more frequently in patients than in controls ( and 0.002; OR = 1.889 and 1.802; 95% CI = 1.153–3.095 and 1.230–2.639, resp.). Conclusions. Polymorphisms in the NRAMP1 gene, VDR gene, HLA-DRB1 gene, and HLA-DQB1 gene are statistically associated with susceptibility to TB in the Chinese Kazakh population. Fang Wu, Wanjiang Zhang, Le Zhang, Jiangdong Wu, Chunzhu Li, Xianjie Meng, Xi Wang, Peng He, and Jie Zhang Copyright © 2013 Fang Wu et al. All rights reserved. Prevalence and Risk Factors of Toxoplasma gondii in Fattening Pigs Farm from Yucatan, Mexico Thu, 06 Jun 2013 14:30:51 +0000 The objective of this study was to estimate the prevalence and identify risk factors associated with the presence of Toxoplasma gondii in pig-fattening farms from Yucatan, Mexico. A cross-sectional study was conducted with a two-stage sampling. There were 429 pigs sampled from 39 farms randomly selected. Blood samples were collected to obtain DNA and serum. The presence of IgM and IgG antibodies was determined by indirect ELISA. Prevalence was estimated by diagnostic test. Potential risk factors to be included in a marginal logistic regression were tested by chi-square or Fisher. The prevalence of IgM and IgG was 92.5% (397/429) (CI 89.9–95.1%) and 95.8% (411/429) (CI 93.7–97.8%), respectively. Regarding PCR, a prevalence of 50.8% (218/429) (CI 45.9–55.6%) was found. The logistic regression showed an association with herd size and type of feeder (). The risk of a case in farms with ≤400 pigs was 27.9 times higher than in farms with >400 pigs. The manual feeder was a significant protective factor associated with the seropositive against T. gondii. Results indicate a high circulation of T. gondii in pig-fattening farms from Yucatan, finding an increased risk of infection for those farms with less than 400 animals and automatic feeders. A. Ortega-Pacheco, K. Y. Acosta Viana, E. Guzmán-Marín, J. C. Segura-Correa, M. Álvarez-Fleites, and M. Jiménez-Coello Copyright © 2013 A. Ortega-Pacheco et al. All rights reserved. A History of the Development of Brucella Vaccines Mon, 03 Jun 2013 13:46:48 +0000 Brucellosis is a worldwide zoonosis affecting animal and human health. In the last several decades, much research has been performed to develop safer Brucella vaccines to control the disease mainly in animals. Till now, no effective human vaccine is available. The aim of this paper is to review and discuss the importance of methodologies used to develop Brucella vaccines in pursuing this challenge. Eric Daniel Avila-Calderón, Ahidé Lopez-Merino, Nammalwar Sriranganathan, Stephen M. Boyle, and Araceli Contreras-Rodríguez Copyright © 2013 Eric Daniel Avila-Calderón et al. All rights reserved. Mixed Production of Filamentous Fungal Spores for Preventing Soil-Transmitted Helminth Zoonoses: A Preliminary Analysis Sun, 21 Apr 2013 16:20:33 +0000 Helminth zoonoses are parasitic infections shared by humans and animals, being the soil-transmitted helminths (STHs) mainly caused by roundworms (ascarids) and hookworms. This study was aimed to assess the individual and/or mixed production of two helminth-antagonistic fungi, one ovicide (Mucor circinelloides) and other predator (Duddingtonia flagrans). Fungi were grown both in Petri plates and in a submerged culture (composed by water, NaCl, Na2HPO4 · 12 H2O, and wheat (Triticum aestivum)). A Fasciola hepatica recombinant protein (FhrAPS) was incorporated to the cultures to improve fungal production. All the cultured plates showed fungal growth, without difference in the development of the fungi when grown alone or mixed. High counts of Mucor spores were produced in liquid media cultures, and no significant differences were achieved regarding single or mixed cultures, or the incorporation of the FhrAPS. A significantly higher production of Duddingtonia spores after the incorporation of the FhrAPS was observed. When analyzing the parasiticide efficacy of the fungal mixture, viability of T. canis eggs reduced to 51%, and the numbers of third stage cyathostomin larvae reduced to 4%. It is concluded, the capability of a fungal mixture containing an ovicide (Mucor) and a predator species (Duddingtonia) for growing together in a submerged medium containing the FhrAPS offers a very interesting tool for preventing STHs. M. S. Arias, C. F. Cazapal-Monteiro, J. Suárez, S. Miguélez, I. Francisco, F. L. Arroyo, J. L. Suárez, A. Paz-Silva, R. Sánchez-Andrade, and P. Mendoza de Gives Copyright © 2013 M. S. Arias et al. All rights reserved. MLST Genotypes and Antibiotic Resistance of Campylobacter spp. Isolated from Poultry in Grenada Sun, 24 Feb 2013 09:18:27 +0000 This study determined whether multilocus sequence types (MLST) of Campylobacter from poultry in 2 farms in Grenada, West Indies, differed by farm, antimicrobial resistance and farm antibiotic use. Farm A used fluoroquinolones in the water and Farm B used tetracyclines. The E-test was used to determine resistance of isolates to seven antibiotics. PCR of the IpxA gene confirmed species and MLST was used to characterize 38 isolates. All isolates were either C. jejuni or C. coli. Farm antibiotic use directly correlated with antimicrobial resistance of Campylobacter isolates. Almost 80% of the isolates from Farm A were fluoroquinolone resistant and 17.9% of the isolates from Farm B were fluoroquinolone resistant. All Campylobacter isolates from Farm A were tetracycline sensitive, whereas 35.7% of isolates from Farm B were tetracycline resistant. Six previously recognized sequence types (STs) and 2 novel STs were identified. Previously recognized STs were those overwhelmingly reported from poultry and humans globally. Isolates with the same ST did not always have the same antibiotic resistance profile. There was little ST overlap between the farms suggesting that within-farm transmission of Campylobacter genotypes may dominate. MLST typing was useful for tracking Campylobacter spp. among poultry units and can help elucidate Campylobacter host-species population structure and its relevance to human health. Diana Stone, Margaret Davis, Katherine Baker, Tom Besser, Rohini Roopnarine, and Ravindra Sharma Copyright © 2013 Diana Stone et al. All rights reserved. Asymptomatic Malaria Correlates with Anaemia in Pregnant Women at Ouagadougou, Burkina Faso Sun, 11 Nov 2012 17:08:01 +0000 Sub-Saharan Africa records each year about thirty-two million pregnant women living in areas of high transmission of Plasmodium falciparum causing malaria. The aim of this study was to carve out the prevalence of asymptomatic malaria among pregnant women and to emphasize its influence on haematological markers. The prevalence of Plasmodium falciparum asymptomatic infection among pregnant women was 30% and 24% with rapid detection test (RDT) and microscopy, respectively. The prevalence of P. falciparum asymptomatic malaria was reduced among pregnant women using sulfadoxine-pyrimethamine's intermittent preventive treatment and 61% of them were anaemic. Anaemia was significantly more common in women infected with P. falciparum compared with the uninfected pregnant women. Most of the women had normal levels of homocysteine and low levels of folate, respectively. Therefore, the systematic diagnosis of malaria should be introduced to pregnant women as a part of the antenatal care. Zoenabo Douamba, Cyrille Bisseye, Florencia W. Djigma, Tegwinde R. Compaoré, Valérie Jean Telesphore Bazie, Virginio Pietra, Jean-Baptiste Nikiema, and Jacques Simpore Copyright © 2012 Zoenabo Douamba et al. All rights reserved. Epithelial Cell Coculture Models for Studying Infectious Diseases: Benefits and Limitations Wed, 05 Oct 2011 09:12:50 +0000 Countless in vitro cell culture models based on the use of epithelial cell types of single lineages have been characterized and have provided insight into the mechanisms of infection for various microbial pathogens. Diverse culture models based on disease-relevant mucosal epithelial cell types derived from gastrointestinal, genitourinary, and pulmonary organ systems have delineated many key host-pathogen interactions that underlie viral, parasitic, and bacterial disease pathogenesis. An alternative to single lineage epithelial cell monoculture, which offers more flexibility and can overcome some of the limitations of epithelial cell culture models based on only single cell types, is coculture of epithelial cells with other host cell types. Various coculture models have been described, which incorporate epithelial cell types in culture combination with a wide range of other cell types including neutrophils, eosinophils, monocytes, and lymphocytes. This paper will summarize current models of epithelial cell coculture and will discuss the benefits and limitations of epithelial cell coculture for studying host-pathogen dynamics in infectious diseases. Benjamin L. Duell, Allan W. Cripps, Mark A. Schembri, and Glen C. Ulett Copyright © 2011 Benjamin L. Duell et al. All rights reserved. A New Microarray System to Detect Streptococcus pneumoniae Serotypes Thu, 09 Jun 2011 15:06:52 +0000 Streptococcus pneumoniae, one of the most common gram-positive pathogens to colonize the human upper respiratory tract, is responsible for many severe infections, including meningitis and bacteremia. A 23-valent pneumococcal vaccine is available to protect against the 23 S. pneumoniae serotypes responsible for 90% of reported bacteremic infections. Unfortunately, current S. pneumoniae serotype testing requires a large panel of expensive antisera, assay results may be subjective, and serotype cross-reactions are common. For this study, we designed an oligonucleotide-based DNA microarray to identify glycosyltransferase gene sequences specific to each vaccine-related serotype. Out of 56 isolates representing different serotypes, only one isolate, representing serotype 23A, was not detected correctly as it could not be distinguished from serotype 23F. Our data suggest that the microarray provides a more cost-effective and reliable way of monitoring pneumococcal capsular types. Yuka Tomita, Akira Okamoto, Keiko Yamada, Testuya Yagi, Yoshinori Hasegawa, and Michio Ohta Copyright © 2011 Yuka Tomita et al. All rights reserved.