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1.  Regulatory Phenotype, PD-1 and TLR3 Expression in T Cells and Monocytes from HCV Patients Undergoing Antiviral Therapy: A Randomized Clinical Trial 
PLoS ONE  2014;9(4):e93620.
Background & Aims
The cellular immunity has a profound impact on the status of hepatitis C virus (HCV) infection. However, the response of cellular immunity on the virological response in patients with antiviral treatment remains largely unclear. We aimed to clarify the response of peripheral T cells and monocytes in chronic hepatitis C patients with antiviral treatment.
Patients with chronic hepatitis C were treated either with interferon alpha-2b plus ribavirin (n = 37) or with pegylated interferon alpha-2a plus ribavirin (n = 33) for up to 24 weeks. Frequencies of peripheral regulatory T-cells (Tregs), programmed death-1 (PD-1) expressing CD4+ T-cells or CD8+ T-cells and toll-like receptor (TLR) 3 expressing CD14+ monocytes were evaluated by flow cytometry in patients at baseline, 12 and 24 weeks following treatment and in 20 healthy controls.
Frequencies of Tregs, PD-1 and TLR3 expressing cells were higher in patients than those in control subjects (P<0.05). Patients with complete early virological response (cEVR) showed lower Tregs, PD-1 expressing CD4+ or CD8+ T-cells than those without cEVR at 12 weeks (P<0.05). Patients with low TLR3 expressing CD14+ monocytes at baseline had a high rate of cEVR (P<0.05).
Low peripheral TLR3 expressing CD14+ monocytes at baseline could serve as a predictor for cEVR of antiviral therapy in chronic HCV-infected patients. The cEVR rates were significantly increased in the patients with reduced circulating Tregs, PD-1 expressing CD4+ or CD8+ T-cells.
Trial Registration
Chinese Clinical Trial Registry ChiCTR10001090.
PMCID: PMC3977904  PMID: 24709775
2.  Cost-Effectiveness of Cranberries vs Antibiotics to Prevent Urinary Tract Infections in Premenopausal Women: A Randomized Clinical Trial 
PLoS ONE  2014;9(4):e91939.
Urinary tract infections (UTIs) are common and result in an enormous economic burden. The increasing prevalence of antibiotic-resistant microorganisms has stimulated interest in non-antibiotic agents to prevent UTIs.
To evaluate the cost-effectiveness of cranberry prophylaxis compared to antibiotic prophylaxis with trimethoprim-sulfamethoxazole (TMP-SMX) over a 12 month period in premenopausal women with recurrent UTIs.
Materials and Methods
An economic evaluation was performed alongside a randomized trial. Primary outcome was the number of UTIs during 12 months. Secondary outcomes included satisfaction and quality of life. Healthcare utilization was measured using questionnaires. Missing data were imputed using multiple imputation. Bootstrapping was used to evaluate the cost-effectiveness of the treatments.
Cranberry prophylaxis was less effective than TMP-SMX prophylaxis, but the differences in clinical outcomes were not statistically significant. Costs after 12 months in the cranberry group were statistically significantly higher than in the TMP-SMX group (mean difference €249, 95% confidence interval 70 to 516). Cost-effectiveness planes and cost-effectiveness acceptability curves showed that cranberry prophylaxis to prevent UTIs is less effective and more expensive than (dominated by) TMP-SMX prophylaxis.
In premenopausal women with recurrent UTIs, cranberry prophylaxis is not cost-effective compared to TMP-SMX prophylaxis. However, it was not possible to take into account costs attributed to increased antibiotic resistance within the framework of this randomized trial; modeling studies are recommended to investigate these costs. Moreover, although we based the dosage of cranberry extract on available evidence, this may not be the optimal dosage. Results may change when this optimal dosage is identified.
Trial Registration ISRCTN50717094
PMCID: PMC3976255  PMID: 24705418
3.  Gut Hormone Pharmacology of a Novel GPR119 Agonist (GSK1292263), Metformin, and Sitagliptin in Type 2 Diabetes Mellitus: Results from Two Randomized Studies 
PLoS ONE  2014;9(4):e92494.
GPR119 receptor agonists improve glucose metabolism and alter gut hormone profiles in animal models and healthy subjects. We therefore investigated the pharmacology of GSK1292263 (GSK263), a selective GPR119 agonist, in two randomized, placebo-controlled studies that enrolled subjects with type 2 diabetes. Study 1 had drug-naive subjects or subjects who had stopped their diabetic medications, and Study 2 had subjects taking metformin. GSK263 was administered as single (25–800 mg; n = 45) or multiple doses (100–600 mg/day for 14 days; n = 96). Placebo and sitagliptin 100 mg/day were administered as comparators. In Study 1, sitagliptin was co-administered with GSK263 or placebo on Day 14 of dosing. Oral glucose and meal challenges were used to assess the effects on plasma glucose, insulin, C-peptide, glucagon, peptide tyrosine-tyrosine (PYY), glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). After 13 days of dosing, GSK263 significantly increased plasma total PYY levels by ∼five-fold compared with placebo, reaching peak concentrations of ∼50 pM after each of the three standardized meals with the 300 mg BID dose. Co-dosing of GSK263 and metformin augmented peak concentrations to ∼100 pM at lunchtime. GSK263 had no effect on active or total GLP-1 or GIP, but co-dosing with metformin increased post-prandial total GLP-1, with little effect on active GLP-1. Sitagliptin increased active GLP-1, but caused a profound suppression of total PYY, GLP-1, and GIP when dosed alone or with GSK263. This suppression of peptides was reduced when sitagliptin was co-dosed with metformin. GSK263 had no significant effect on circulating glucose, insulin, C-peptide or glucagon levels. We conclude that GSK263 did not improve glucose control in type 2 diabetics, but it had profound effects on circulating PYY. The gut hormone effects of this GPR119 agonist were modulated when co-dosed with metformin and sitagliptin. Metformin may modulate negative feedback loops controlling the secretion of enteroendocrine peptides.
Trial Registration: NCT01119846 NCT01128621
PMCID: PMC3974707  PMID: 24699248
4.  Neuro-Intensive Treatment Targeting Intracranial Hypertension Improves Outcome in Severe Bacterial Meningitis: An Intervention-Control Study 
PLoS ONE  2014;9(3):e91976.
To evaluate the efficacy of early intracranial pressure (ICP)-targeted treatment, compared to standard intensive care, in adults with community acquired acute bacterial meningitis (ABM) and severely impaired consciousness.
A prospectively designed intervention-control comparison study of adult cases from September 2004 to January 2012.
Included patients were confirmed ABM-cases, aged 16–75 years, with severely impaired mental status on admission. Fifty-two patients, given ICP-targeted treatment at the neuro-intensive care unit, and 53 control cases, treated with conventional intensive care, were included. All the patients received intensive care with mechanical ventilation, sedation, antibiotics and corticosteroids according to current guidelines. Additional ICP-treatment in the intervention group included cerebrospinal fluid drainage using external ventricular catheters (n = 48), osmotherapy (n = 21), hyperventilation (n = 13), external cooling (n = 9), gram-doses of methylprednisolone (n = 3) and deep barbiturate sedation (n = 2) aiming at ICP <20 mmHg and a cerebral perfusion pressure of >50 mmHg.
The primary endpoint was mortality at two months and secondary endpoint was Glasgow outcome score and hearing ability at follow-up at 2–6 months.
The mortality was significantly lower in the intervention group compared to controls, 5/52 (10%) versus 16/53 (30%; relative risk reduction 68%; p<0.05). Furthermore, only 17 patients (32%) in the control group fully recovered compared to 28 (54%) in the intervention group (relative risk reduction 40%; p<0.05).
Early neuro-intensive care using ICP-targeted therapy, mainly cerebrospinal fluid drainage, reduces mortality and improves the overall outcome in adult patients with ABM and severely impaired mental status on admission.
PMCID: PMC3965390  PMID: 24667767
5.  In Vivo and In Vitro Studies of Th17 Response to Specific Immunotherapy in House Dust Mite-Induced Allergic Rhinitis Patients 
PLoS ONE  2014;9(3):e91950.
T helper (Th)17 cells have been implicated in the development of allergic rhinitis (AR), but their response to specific immunotherapy (SIT) remains unclear. We investigated the impact of SIT on Th17 response and Th1/Th2 changes in AR patients. Blood samples from AR patients (n = 20) who were monosensitized to house dust mite (HDM) were collected before the initiation of SIT (SIT-untreated) and after the end of 2-year SIT (SIT-treated) treatment. Twenty healthy volunteers were recruited as controls. In vitro HDM stimulation in peripheral blood mononuclear cells (PBMCs) was also performed. Expression levels of Th17 associated genes were determined in both PBMCs and plasma by PCR and ELISA, while Th17/Th1/Th2/IL10 producing cell proportions were evaluated in PBMCs by flow cytometry. The SIT effect was evaluated by assessing clinical symptoms. mRNA levels of Th17 specific genes (IL17 and RORC) were increased in SIT-untreated AR versus controls, and decreased following SIT treatment. SIT can change the production of Th17 associated genes (reduction of IL17, IL6, and IL23, but increase of IL27) in plasma from AR patients. Th2/Th1 ratio and proportions of Th17 cells were suppressed while IL10 producing CD4+ T cells were elevated after SIT. In vitro HDM challenge presents concordant patterns with in vivo findings: 1) increase of Th2 and Th17 response in AR patients; 2) suppression of IL10 producing CD4+ T cells in SIT-untreated AR but elevation in SIT-treated AR patients. Most importantly, a positive correlation between IL17 mRNA/protein levels and clinical symptom scores was observed. SIT significantly inhibits Th17 mediated inflammation in AR and IL17 may be a useful biomarker for both AR severity and SIT therapeutic effect.
Trial Registration
Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12613000445774
PMCID: PMC3960160  PMID: 24647473
6.  Comparison between the Therapeutic Effect of Metformin, Glimepiride and Their Combination as an Add-On Treatment to Insulin Glargine in Uncontrolled Patients with Type 2 Diabetes 
PLoS ONE  2014;9(3):e87799.
To compare the commonly prescribed oral anti-diabetic drug (OAD) combinations to use as an add-on therapy with insulin glargine in patients with uncontrolled type 2 diabetes despite submaximal doses of OADs.
People with inadequately controlled type 2 diabetes (n = 99) were randomly assigned on a 1∶1∶1 basis to receive insulin glargin, with fixed doses of glimepiride, metformin, and glimepiride plus metformin. Outcomes assessed included HbA1c, the changes in fasting glucose levels, body weight, serum lipids values, insulin dose and symptomatic hypoglycemia.
After 24 weeks, HbA1C levels improved from (mean ± SD) 8.5±0.9% to 7.7±0.8% (69.0±10.0 mmol/mol to 60.8±8.6 mmol/mol) with insulin glargine plus metformin, from 8.4±1.0% to 7.7±1.3% (68.8±10.6 mmol/mol to 61.1±14.4 mmol/mol) with insulin glargine plus glimepiride and from 8.7±0.9% to 7.3±0.6% (71.7±9.8 mmol/mol to 56.2±6.7 mmol/mol) with insulin glargine plus glimepirde plus metformin. The decrease in HbA1c was more pronounced with insulin glargine plus glimepiride plus metformin than with insulin glargine plus metformin (0.49% [CI, 0.16% to 0.82%]; P = 0.005) (5.10 mmol/mol [CI, 1.64 to 8.61]; P = 0.005) and insulin glargine plus glimepiride (0.59% [CI, 0.13% to 1.05%]; P = 0.012) (5.87 mmol/mol [CI, 1.10 to 10.64]; P = 0.012) (overall P = 0.02). Weight gain and the risk of hypoglycemia of any type did not significantly differ among the treatment groups.
The combination therapy of metformin and glimepiride plus glargine insulin resulted in a significant improvement in overall glycemic control as compared with the other combinations.
Trial registration information, NCT00708578.
The approval number of Kangbuk Samsung hospital's institutional review board (IRB): C0825.
PMCID: PMC3948620  PMID: 24614911
7.  Maternal Valacyclovir and Infant Cytomegalovirus Acquisition: A Randomized Controlled Trial among HIV-Infected Women 
PLoS ONE  2014;9(2):e87855.
Studies in HIV-1-infected infants and HIV-1-exposed, uninfected infants link early cytomegalovirus (CMV) acquisition with growth delay and cognitive impairment. We investigated maternal valacyclovir to delay infant acquisition of CMV.
Pregnant women with HIV-1, HSV-2 and CD4 count >250 cells/µl were randomized at 34 weeks gestation to 500 mg twice-daily valacyclovir or placebo for 12 months. Maternal CMV DNA was measured in plasma at 34 weeks gestation, in cervical secretions at 34 and 38 weeks gestation, and in breast milk at 7 postpartum timepoints; infant CMV DNA was measured in dried blood spots at 8 timepoints including birth.
Among 148 women, 141 infants were compared in intent-to-treat analyses. Maternal and infant characteristics were similar between study arms. Infant CMV acquisition did not differ between study arms, with 46/70 infants (66%) in placebo arm and 47/71 infants (66%) in the valacyclovir arm acquiring CMV; median time to CMV detection did not differ. CMV DNA was detected in 92% of 542 breast milk specimens with no difference in CMV level between study arms. Change in cervical shedding of CMV DNA between baseline and 38 weeks was 0.40-log greater in the placebo arm than the valacyclovir arm (p = 0.05).
In this cohort of HIV-1-seropositive mothers, two-thirds of infants acquired CMV by one year. Maternal valacyclovir had no effect on timing of infant CMV acquisition or breast milk CMV viral loads, although it modestly reduced cervical CMV shedding. Maternal prophylaxis to reduce infant CMV acquisition warrants further evaluation in trials with antiviral agents.
Trials Registration NCT00530777
PMCID: PMC3913686  PMID: 24504006
8.  The Value of Serum Prealbumin in the Diagnosis and Therapeutic Response of Tuberculosis: A Retrospective Study 
PLoS ONE  2013;8(11):e79940.
The aim of this study was to examine serum prealbumin (PA) levels in patients with tuberculosis and lung cancer, and to evaluate the correlations of serum PA levels with clinicopathological characteristics.
Total 760 patients were included in the study: 320 patients with tuberculosis, 320 patients with lung cancer, and 120 healthy subjects. Serum PA was detected using a biochemical analyzer to determine the value of serum PA in the diagnosis and therapeutic response of tuberculosis.
Compared to lung cancer and healthy individuals, TB patients were more frequent in suffering from low serum PA (75.0% vs.30.9% vs.6.7%,P<0.01), and the serum PA levels of TB patients were significantly reduced (137.5±42.4 mg/L vs. 183.5±49.1 mg/L vs. 240.0±43.9 mg/L, P<0.01). Among various clinical characteristics, type (with pleuritis), age (≥60), ESR (>20 mm/h) and smoking status (≥20 pack×years) were associated with low serum PA levels of TB patients, while ECOG performance status (≥2) was associated with low serum PA levels of lung cancer patients. The change of serum PA levels was in accordance with the therapeutic effects of anti-TB drugs, which might present a valuable and objective indicator for monitoring the therapeutic effects of TB drugs on TB patients.
Low serum prealbumin levels are very common in TB patients and can be served as a potential indicator for differential diagnosis of lung cancer and monitoring the therapeutic effects of TB drugs.
PMCID: PMC3833965  PMID: 24260323
9.  Combination of Cytokine Responses Indicative of Latent TB and Active TB in Malawian Adults 
PLoS ONE  2013;8(11):e79742.
An IFN-γ response to M. tuberculosis-specific antigens is an effective biomarker for M. tuberculosis infection but it cannot discriminate between latent TB infection and active TB disease. Combining a number of cytokine/chemokine responses to M. tuberculosis antigens may enable differentiation of latent TB from active disease.
Asymptomatic recently-exposed individuals (spouses of TB patients) were recruited and tuberculin skin tested, bled and followed-up for two years. Culture supernatants, from a six-day culture of diluted whole blood samples stimulated with M. tuberculosis-derived PPD or ESAT-6, were measured for IFN-γ, IL-10, IL-13, IL-17, TNF-α and CXCL10 using cytokine ELISAs. In addition, 15 patients with sputum smear-positive pulmonary TB were recruited and tested.
Spouses with positive IFN-γ responses to M. tuberculosis ESAT-6 (>62.5 pg/mL) and TB patients showed high production of IL-17, CXCL10 and TNF-α. Higher production of IL-10 and IL-17 in response to ESAT-6 was observed in the spouses compared with TB patients while the ratios of IFN-γ/IL-10 and IFN-γ/IL-17 in response to M. tuberculosis-derived PPD were significantly higher in TB patients compared with the spouses. Tuberculin skin test results did not correlate with cytokine responses.
CXCL10 and TNF-α may be used as adjunct markers alongside an IFN-γ release assay to diagnose M. tuberculosis infection, and IL-17 and IL-10 production may differentiate individuals with LTBI from active TB.
PMCID: PMC3832606  PMID: 24260295
10.  Identification of Novel sRNAs in Mycobacterial Species 
PLoS ONE  2013;8(11):e79411.
Bacterial small RNAs (sRNAs) are short transcripts that typically do not encode proteins and often act as regulators of gene expression through a variety of mechanisms. Regulatory sRNAs have been identified in many species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. Here, we use a computational algorithm to predict sRNA candidates in the mycobacterial species M. smegmatis and M. bovis BCG and confirmed the expression of many sRNAs using Northern blotting. Thus, we have identified 17 and 23 novel sRNAs in M. smegmatis and M. bovis BCG, respectively. We have also applied a high-throughput technique (Deep-RACE) to map the 5′ and 3′ ends of many of these sRNAs and identified potential regulators of sRNAs by analysis of existing ChIP-seq datasets. The sRNAs identified in this work likely contribute to the unique biology of mycobacteria.
PMCID: PMC3828370  PMID: 24244498
11.  Severe Painful Vaso-Occlusive Crises and Mortality in a Contemporary Adult Sickle Cell Anemia Cohort Study 
PLoS ONE  2013;8(11):e79923.
Frequent painful vaso-occlusive crises (VOCs) were associated with mortality in the Cooperative Study of Sickle Cell Disease (CSSCD) over twenty years ago. Modern therapies for sickle cell anemia (SCA) like hydroxyurea are believed to have improved overall patient survival. The current study sought to determine the relevance of the association between more frequent VOCs and death and its relative impact upon overall mortality compared to other known risk factors in a contemporary adult SCA cohort.
Two hundred sixty four SCA adults were assigned into two groups based on patient reported outcomes for emergency department (ED) visits or hospitalizations for painful VOC treatment during the 12 months prior to evaluation.
Higher baseline hematocrit (p = 0.0008), ferritin (p = 0.005), and HDL cholesterol (p = 0.01) were independently associated with 1 or more painful VOCs requiring an ED visit or hospitalization for acute pain. During a median follow-up of 5 years, mortality was higher in the ED visit/hospitalization group (relative risk [RR] 2.68, 95% CI 1.1-6.5, p = 0.03). Higher tricuspid regurgitatant jet velocity (TRV) (RR 2.41, 95% CI 1.5-3.9, p < 0.0001), elevated ferritin (RR 4.00, 95% CI 1.8-9.0, p = 0.001) and lower glomerular filtration rate (RR=2.73, 95% CI 1.6-4.6, p < 0.0001) were also independent risk factors for mortality.
Severe painful VOCs remain a marker for SCA disease severity and premature mortality in a modern cohort along with other known risk factors for death including high TRV, high ferritin and lower renal function. The number of patient reported pain crises requiring healthcare utilization is an easily obtained outcome that could help to identify high risk patients for disease modifying therapies.
Trial Registration NCT00011648
PMCID: PMC3818240  PMID: 24224021
12.  Changes of Cytokines during a Spaceflight Analog - a 45-Day Head-Down Bed Rest 
PLoS ONE  2013;8(10):e77401.
Spaceflight is associated with deregulation in the immune system. Head-down bed rest (HDBR) at -6° is believed to be the most practical model for examining multi-system responses to microgravity in humans during spaceflight. In the present study, a 45-day HDBR was performed to investigate the alterations in human immune cell distributions and their functions in response to various stimuli. The effect of countermeasure, Rhodiola rosea (RR) treatment, was also examined. A significant decrease of interferon-γ (IFN-γ) and interleukin-17 (IL-17) productions by activated T cells, increase of IL-1β and IL-18 by activated B and myeloid cells were observed during HDBR. The upregulation of serum cortisol was correlated with the changes of IL-1 family cytokines. In addition, a significant increase of memory T and B cell and regulatory T cells (Treg) were also detected. The uptake of RR further decreased IFN-γ level and slowed down the upregulation of IL-1 family cytokines. These data suggest that for prolonged HDBR and spaceflight, the decreased protective T cell immunity and enhanced proinflammatory cytokines should be closely monitored. The treatment with RR may play an important role in suppressing proinflammatory cytokines but not in boosting protective T cell immunity.
PMCID: PMC3797033  PMID: 24143230
13.  Development of a New DNA Vaccine for Alzheimer Disease Targeting a Wide Range of Aβ Species and Amyloidogenic Peptides 
PLoS ONE  2013;8(9):e75203.
It has recently been determined that not only Aβ oligomers, but also other Aβ species and amyloidogenic peptides are neurotoxic in Alzheimer disease (AD) and play a pivotal role in AD pathogenesis. In the present study, we attempted to develop new DNA vaccines targeting a wide range of Aβ species. For this purpose, we first performed in vitro assays with newly developed vaccines to evaluate Aβ production and Aβ secretion abilities and then chose an IgL-Aβx4-Fc-IL-4 vaccine (designated YM3711) for further studies. YM3711 was vaccinated to mice, rabbits and monkeys to evaluate anti-Aβ species antibody-producing ability and Aβ reduction effects. It was found that YM3711 vaccination induced significantly higher levels of antibodies not only to Aβ1-42 but also to AD-related molecules including AβpE3-42, Aβ oligomers and Aβ fibrils. Importantly, YM3711 significantly reduced these Aβ species in the brain of model mice. Binding and competition assays using translated YM3711 protein products clearly demonstrated that a large part of antibodies induced by YM3711 vaccination are directed at conformational epitopes of the Aβ complex and oligomers. Taken together, we demonstrate that YM3711 is a powerful DNA vaccine targeting a wide range of AD-related molecules and is worth examining in preclinical and clinical trials.
PMCID: PMC3785508  PMID: 24086465
14.  Association of dietary fat intakes with risk of esophageal and gastric cancer in the NIH-AARP Diet and Health study 
The aim of this study was to investigate whether intakes of total fat and fat subtypes were associated with esophageal adenocarcinoma (EAC), esophageal squamous cell carcinoma (ESCC), gastric cardia or gastric non-cardia adenocarcinoma. From 1995–1996, dietary intake data was reported by 494,978 participants of the NIH-AARP cohort. 630 EAC, 215 ESCC, 454 gastric cardia and 501 gastric non-cardia adenocarcinomas accrued to the cohort. Cox proportional hazards regression was used to examine the association between the dietary fat intakes, whilst adjusting for potential confounders. Though apparent associations were observed in energy-adjusted models, multivariate adjustment attenuated results to null (e.g. EAC energy adjusted hazard ratio (HR) and 95% confidence interval (95%CI) 1.66 (1.27–2.18) P for trend <0.01; EAC multivariate adjusted HR (95%CI) 1.17 (0.84–1.64) P for trend=0.58). Similar patterns were also observed for fat subtypes (e.g. EAC saturated fat, energy adjusted HR (95%CI) 1.79 (1.37–2.33) P for trend <0.01; EAC saturated fat, multivariate adjusted HR (95%CI) 1.27 (0.91–1.78) P for trend=0.28). However, in multivariate models an inverse association for polyunsaturated fat (continuous) was seen for EAC in subjects with a body mass index (BMI) in the normal range (18.5–<25 kg/m2) (HR (95%CI) 0.76 (0.63–0.92)), that was not present in overweight subjects (HR (95%CI) 1.04 (0.96–1.14)), or in unstratified analysis (HR (95%CI) 0.97 (0.90–1.05)). P for interaction=0.02. Overall, we found null associations between the dietary fat intakes with esophageal or gastric cancer risk; though a protective effect of polyunsaturated fat intake was seen for EAC in subjects with a normal BMI.
PMCID: PMC3346853  PMID: 22116732
cohort; dietary fat; esophageal neoplasms; stomach neoplasms; prospective
15.  Two-Step Tuberculin Skin Testing in School-Going Adolescents with Initial 0-4 Millimeter Responses in a High Tuberculosis Prevalence Setting in South India 
PLoS ONE  2013;8(9):e71470.
The utility of two-step tuberculin skin testing among adolescents in high tuberculosis prevalence settings is not well established.
To determine the proportion and determinants of a 0-4 mm response to an initial standard tuberculin skin test (TST) and evaluating 'boosting' with repeat testing.
Adolescents between 11 and 18 years attending schools/colleges underwent a TST; those with a response of between 0–4 mm had a repeat TST 1-4 weeks later.
Initial TST was done for 6608/6643 participants; 1257 (19%) developed a 0-4 mm response to the initial TST. Younger age and under-nutrition were more likely to be associated with a 0-4 mm response, while the presence of BCG (Bacillus Calmette Guerin) scar and higher socio-economic class were less likely to be associated with a 0-4 mm response. On repeat testing boosting was seen in 13.2% (145/1098; ≥ 6 mm over the initial test) while 4.3% showed boosting using a more conservative cutoff of a repeat TST ≥ 10 mm with an increment of at least 6 mm (47/1098). History of exposure to a tuberculosis (TB) case was associated with enhanced response.
The proportion of adolescents who demonstrated boosting on two-step TST testing in our study was relatively low. As a result repeat testing did not greatly alter the prevalence of TST positivity. However, the two-step TST helps identify individuals who can potentially boost their immune response to a second test, and thus, prevents them from being misclassified as those with newly acquired infection, or tuberculin converters. While two-step tuberculin skin testing may have a limited role in population- level TST surveys, it may be useful where serial tuberculin testing needs to be performed to distinguish those who show an enhanced response or boosters from those who indeed have a new infection, or converters.
PMCID: PMC3765300  PMID: 24039716
16.  Clinical Trial to Evaluate the Safety and Immunogenicity of a Trivalent Surface Antigen Seasonal Influenza Vaccine Produced in Mammalian Cell Culture and Administered to Young and Elderly Adults with and without A(H1N1) Pre-Vaccination 
PLoS ONE  2013;8(8):e70866.
Vaccination against influenza is an important means of reducing morbidity and mortality in subjects at risk. The prevalent viral strains responsible for seasonal epidemics usually change annually, but the WHO recommendations for the 2011/2012-season in the Northern hemisphere included the same antigens as for the previous season.
We conducted a single-center, single-arm study involving 62 younger (18–60 years) and 64 older (>60 years) adults to test the immunogenicity, safety and tolerability of a trivalent surface antigen, inactivated influenza vaccine produced in mammalian cell-culture. The vaccine contained 15 µg hemagglutinin of each of the virus strains recommended for the 2011–2012 Northern hemisphere winter season (A/California/7/09 (H1N1)-; A/Perth/16/09 (H3N2)-; B/Brisbane/60/08-like strain) in a non-adjuvanted preservative-free formulation. Antibody response was measured by hemagglutination inhibition 21 days after immunization. Adverse events and safety were assessed using subject diary cards and telephone interviews.
Seroconversion or a 4-fold antibody increase in antibody titers was detectable against A(H1N1) in 68% of both younger and older adults, against A(H3N2) in 53% and 27%, and against the B influenza strain in 35% and 17%. Antibody titers of 40 or more were observed against A(H1N1) in 87% and 90% of younger and older adults, against A(H3N2) in 98% and 98%, and against the B influenza strain in 93% and 90%. Pre-vaccination antibody titers were protective against A(H1N1), A(H3N2) and B in 38%, 58% and 58%, respectively, of younger and in 43%, 88% and 70% of older adults. Among subjects with previous A(H1N1) vaccination only 48% of younger and 47% of older adults had protective A(H1N1) antibodies at inclusion. Adverse reactions were generally mild. The most frequently reported reactions were pain at the injection site, myalgia and fatigue.
The vaccine generated protective antibodies against all three viral strains and had an acceptable safety profile in both younger and older adults.
Trial Registration NCT01422512
PMCID: PMC3745456  PMID: 23976960
17.  Neutral and Acidic Oligosaccharides Supplementation Does Not Increase the Vaccine Antibody Response in Preterm Infants in a Randomized Clinical Trial 
PLoS ONE  2013;8(8):e70904.
In preterm infants, a decreased immunological response and lower serological effectiveness are observed after immunizations due to ineffectiveness of both humoral and cellular immune mechanisms.
To determine the effect of 80% neutral oligosaccharides [small-chain galacto-oligosaccharides/long-chain fructo-oligosaccharides (scGOS/lcFOS)] in combination with 20% pectin-derived acidic oligosaccharides (pAOS) on antibody concentrations after DTaP-IPV-Hib immunization in preterm infants.
In this randomized clinical trial, preterm infants with gestational age <32 weeks and/or birth weight <1500 g received enteral supplementation with scGOS/lcFOS/pAOS or placebo (maltodextrin) between days 3 and 30 of life. Blood samples were collected at 5 and 12 months of age.
In total, 113 infants were included. Baseline and nutritional characteristics were not different in both groups. Geometric mean titers were not different after prebiotic supplementation at 5 months, Ptx (37/44 EU/ml), FHA (78/96 EU/ml), Prn (78/80 EU/ml), Diphtheria (0.40/0.57 IU/ml), Tetanus (0.74/0.99 IU/ml) and Hib (0.35/0.63 µg/ml), and at 12 months Ptx (55/66 EU/ml), FHA (122/119 EU/ml), Prn (116/106 Eu/ml), Diphtheria (0.88/1.11 IU/ml), Tetanus (1.64/1.79 IU/ml) and Hib (2.91/2.55 µg/ml).
Enteral supplementation of neutral (scGOS/lcFOS) and acidic oligosaccharides (pAOS) does not improve the immunization response in preterm infants.
Trial Registration ISRCTN16211826 ISRCTN16211826
PMCID: PMC3738516  PMID: 23951035
18.  Cost Effectiveness of Cryptococcal Antigen Screening as a Strategy to Prevent HIV-Associated Cryptococcal Meningitis in South Africa 
PLoS ONE  2013;8(7):e69288.
Cryptococcal meningitis (CM)-related mortality may be prevented by screening patients for sub-clinical cryptococcal antigenaemia (CRAG) at antiretroviral-therapy (ART) initiation and pre-emptively treating those testing positive. Prior to programmatic implementation in South Africa we performed a cost-effectiveness analysis of alternative preventive strategies for CM.
Cost-effectiveness analysis.
Using South African data we modelled the cost-effectiveness of four strategies for patients with CD4 cell-counts <100 cells/µl starting ART 1) no screening or prophylaxis (standard of care), 2) universal primary fluconazole prophylaxis, 3) CRAG screening with fluconazole treatment if antigen-positive, 4) CRAG screening with lumbar puncture if antigen-positive and either amphotericin-B for those with CNS disease or fluconazole for those without. Analysis was limited to the first year of ART.
The least costly strategy was CRAG screening followed by high-dose fluconazole treatment of all CRAG-positive individuals. This strategy dominated the standard of care at CRAG prevalence ≥0.6%. Although CRAG screening followed by lumbar puncture in all antigen-positive individuals was the most effective strategy clinically, the incremental benefit of LPs and amphotericin therapy for those with CNS disease was small and additional costs were large (US$158 versus US$51per person year; incremental cost effectiveness ratio(ICER) US$889,267 per life year gained). Both CRAG screening strategies are less costly and more clinically effective than current practice. Primary prophylaxis is more effective than current practice, but relatively cost-ineffective (ICER US$20,495).
CRAG screening would be a cost-effective strategy to prevent CM-related mortality among patients initiating ART in South Africa. These findings provide further justification for programmatic implementation of CRAG screening.
PMCID: PMC3716603  PMID: 23894442
19.  Intracellular Delivery of Lipopolysaccharide Induces Effective Th1-Immune Responses Independent of IL-12 
PLoS ONE  2013;8(7):e68671.
Lipopolysaccharide (LPS) is responsible for many of the inflammatory responses and pathogenic effects of Gram-negative bacteria, however, it also induces protective immune responses. LPS induces the production of inflammatory cytokines such as TNF-α, IL-6, and IL-12 from dendritic cells (DCs) and macrophages. It is thought that IL-12 is required for one of the protective immune responses induced by LPS, the T helper 1 (Th1)-immune response, which include the production of IFN-γ from Th1cells and IgG2c class switching. Here, we clearly demonstrate that intracellular delivery of LPS by LPS-formulated liposomes (LPS-liposomes) does not induce the production of inflammatory cytokines from DCs, but enhances Th1-immune responses via type-I IFNs, independent of IL-12. Collectively, our results strongly suggest that LPS-liposomes can effectively induce Th1-immune responses without inducing unnecessary inflammation, and may be useful as an immune adjuvant to induce protective immunity.
PMCID: PMC3714268  PMID: 23874715
20.  Triglyceride/HDL Ratio as a Screening Tool for Predicting Success at Reducing Anti-Diabetic Medications Following Weight Loss 
PLoS ONE  2013;8(7):e69285.
Background and Objectives
Intentional weight loss, by reducing insulin resistance, results in both better glycemic control and decreased need for anti-diabetic medications. However, not everyone who is successful with weight loss is able to reduce anti-diabetic medication use. In this retrospective cohort study, we assessed the predictive accuracy of baseline triglyceride (TGL)/HDL ratio, a marker of insulin resistance, to screen patients for success in reducing anti-diabetic medication use with weight loss.
Case records of 121 overweight and obese attendees at two outpatient weight management centers were analyzed. The weight loss intervention consisted of a calorie-restricted diet (~1000Kcal/day deficit), a behavior modification plan, and a plan for increasing physical activity.
Mean period of follow-up was 12.5 ± 3.5 months. By study exit, mean weight loss and mean HbA1c% reduction were 15.4 ± 5.5 kgs and 0.5 ± 0.2% respectively. 81 (67%) in the study cohort achieved at least 1 dose reduction of any anti-diabetic medication. Tests for predictive accuracy of baseline TGL/HDL ratio ≤ 3 to determine success with dose reductions of anti-diabetic medications showed a sensitivity, specificity, positive predictive value, negative predictive value, area under the curve, likelihood ratio (LR) + and LR-of 81, 83, 90, 70, 78, 4.8 and 0.2, respectively. Reproducibility of TGL/HDL ratio was acceptable.
TGL/HDL ratio shows promise as an effective screening tool to determine success with dose reductions of anti-diabetic medications. The results of our study may inform the conduct of a systematic review using data from prior weight loss trials.
PMCID: PMC3712020  PMID: 23869240
21.  Randomized Clinical Trial of Thrice-Weekly 4-Month Moxifloxacin or Gatifloxacin Containing Regimens in the Treatment of New Sputum Positive Pulmonary Tuberculosis Patients 
PLoS ONE  2013;8(7):e67030.
Shortening tuberculosis (TB) treatment duration is a research priority. This paper presents data from a prematurely terminated randomized clinical trial, of 4-month moxifloxacin or gatifloxacin regimens, in South India.
Newly diagnosed, sputum-positive HIV-negative pulmonary TB patients were randomly allocated to receive gatifloxacin or moxifloxacin, along with isoniazid and rifampicin for 4 months with pyrazinamide for first 2 months (G or M) or isoniazid and rifampicin for 6 months with ethambutol and pyrazinamide for first 2 months (C). All regimens were administered thrice-weekly. Clinical and bacteriological assessments were done monthly during treatment and for 24 months post-treatment. The Data and Safety Monitoring Board recommended termination of the trial due to high TB recurrence rates in the G and M regimens.
Of 416 patients in intent-to-treat analysis, 6 (5%) of 124, 2 (2%) of 110 and 2 (2%) of 137 patients with drug-susceptible TB in the G, M and C arms respectively had unfavorable response at the end of treatment; during the next 24 months, 17 (15%) of 115, 11 (11%) of 104 and 8 (6%) of 132 patients respectively, had TB recurrence. Of 38 drug-resistant patients 1 of 8 and 3 of 26 in the G and C arms respectively had unfavourable response at the end of treatment; and TB recurrence occurred in 2 of 7 and 2 of 23 patients, respectively. The differences in TB recurrence rates between the G and C arms was statistically significant (p = 0.02). Gastro-intestinal symptoms occurred in 23%, 22% and 9% of patients in the G, M and C arms respectively, but most reactions were mild and manageable with symptomatic measures; 1% required regimen modification.
4-month thrice-weekly regimens of gatifloxacin or moxifloxacin with isoniazid, rifampicin and pyrazinamide, were inferior to standard 6-month treatment, in patients with newly diagnosed sputum positive pulmonary TB.
Trial Registration
Clinical Trials Registry of India CTRI/2012/10/003060
PMCID: PMC3700922  PMID: 23843980
22.  Rotavirus Vaccination Coverage among Children Aged 2-59 Months: A Report from Guangzhou, China 
PLoS ONE  2013;8(6):e68169.
We aimed to estimate the Lanzhou lamb rotavirus (LLR) vaccination coverage (VC) and timeliness among children aged 2 to 59 months in Guangzhou, China.
An electronic system-based VC survey was conducted using stratified cluster random sampling.
We reported an overall Lanzhou lamb rotavirus vaccine coverage of 25.3% among children aged 2-59 months (2-8 months, 2.6%) in Guangzhou, China.
Great efforts should be taken to increase LLR VC in eligible children in Guangzhou, China.
PMCID: PMC3695951  PMID: 23840828
23.  Parasitic infection may be associated with discordant responses to QuantiFERON and tuberculin skin test in apparently healthy children and adolescents in a tuberculosis endemic setting, Ethiopia 
BMC Infectious Diseases  2013;13:265.
M. tuberculosis remains one of the world’s deadliest pathogens in part because of its ability to establish persistent, latent infections, which can later reactivate to cause disease. In regions of the globe where disease is endemic, as much as 50% of the population is thought to be latently infected, complicating diagnosis and tuberculosis control. The tools most commonly used for diagnosis of latent M. tuberculosis infection are the tuberculin skin test and the newer interferon-gamma release assays, both of which rely on an antigen-specific memory response as an indicator of infection. It is clear that the two tests, do not always give concordant results, but the factors leading to this are only partially understood.
In this study we examined 245 healthy school children aged from 12 to 20 years from Addis Ababa, a tuberculosis-endemic region, characterised them with regard to response in the tuberculin skin test and QuantIFERON™ test and assessed factors that might contribute to discordant responses.
Although concordance between the tests was generally fair (90% concordance), there was a subset of children who had a positive QuantIFERON™ result but a negative tuberculin skin test. After analysis of multiple parameters the data suggest that discordance was most strongly associated with the presence of parasites in the stool.
Parasitic gut infections are frequent in most regions where M. tuberculosis is endemic. This study, while preliminary, suggests that the tuberculin skin test should be interpreted with caution where this may be the case.
PMCID: PMC3674899  PMID: 23738853
Latent tuberculosis; QuantiFERON; TST; Adolescents; Children; Parasites
24.  Serum Interleukin-18 at Commencement of Renal Replacement Therapy Predicts Short-Term Prognosis in Critically Ill Patients with Acute Kidney Injury 
PLoS ONE  2013;8(5):e66028.
Acute kidney injury (AKI) requiring renal replacement therapy (RRT) in critically ill patients results in a high hospital mortality. Outcome prediction in this selected high-risk collective is challenging due to the lack of appropriate biomarkers. The aim of this study was to identify outcome-specific biomarkers in this patient population.
Methodology/Principal Findings
Serum samples were collected from 101 critically ill patients with AKI at the initiation of RRT in intensive care units (ICUs) of a tertiary care university hospital between August 2008 and March 2011. Measurements of serum levels of cystatin C (CysC), neutrophil gelatinase-associated lipocalin, and interleukin-18 (IL-18) were performed. The primary outcome measure was hospital mortality. The observed overall mortality rate was 56.4% (57/101). Multiple logistic regression analysis indicated that the serum IL-18 and CysC concentrations and Acute Physiology and Chronic Health Evaluation III (ACPACHE III) scores determined on the first day of RRT were independent predictors of hospital mortality. The APACHE III score had the best discriminatory power (0.872±0.041, p<0.001), whereas serum IL-18 had the best Youden index (0.65) and the highest correctness of prediction (83%). Cumulative survival rates at 6-month follow-up following hospital discharge differed significantly (p<0.001) for serum IL-18 <1786 pg/ml vs. ≥1786 pg/ml in these critically ill patients.
In this study, we confirmed the grave prognosis for critically ill patients at the commencement of RRT and found a strong correlation between serum IL-18 and the hospital mortality of ICU patients with dialysis-dependent AKI. In addition, we demonstrated that the APACHE III score has the best discriminative power for predicting hospital mortality in these critically ill patients.
PMCID: PMC3669263  PMID: 23741523
25.  Using Magnetic Resonance Imaging to Evaluate Dendritic Cell-Based Vaccination 
PLoS ONE  2013;8(5):e65318.
Cancer immunotherapy with antigen-loaded dendritic cell-based vaccines can induce clinical responses in some patients, but further optimization is required to unlock the full potential of this strategy in the clinic. Optimization is dependent on being able to monitor the cellular events that take place once the dendritic cells have been injected in vivo, and to establish whether antigen-specific immune responses to the tumour have been induced. Here we describe the use of magnetic resonance imaging (MRI) as a simple, non-invasive approach to evaluate vaccine success. By loading the dendritic cells with highly magnetic iron nanoparticles it is possible to assess whether the injected cells drain to the lymph nodes. It is also possible to establish whether an antigen-specific response is initiated by assessing migration of successive rounds of antigen-loaded dendritic cells; in the face of a successfully primed cytotoxic response, the bulk of antigen-loaded cells are eradicated on-route to the node, whereas cells without antigen can reach the node unchecked. It is also possible to verify the induction of a vaccine-induced response by simply monitoring increases in draining lymph node size as a consequence of vaccine-induced lymphocyte trapping, which is an antigen-specific response that becomes more pronounced with repeated vaccination. Overall, these MRI techniques can provide useful early feedback on vaccination strategies, and could also be used in decision making to select responders from non-responders early in therapy.
PMCID: PMC3667033  PMID: 23734246

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