Viral Co-Infections in Pediatric Patients Hospitalized with Lower Tract Acute Respiratory Infections

Background

Molecular techniques can often reveal a broader range of pathogens in respiratory infections. We aim to investigate the prevalence and age pattern of viral co-infection in children hospitalized with lower tract acute respiratory infection (LT-ARI), using molecular techniques.

Methods

A nested polymerase chain reaction approach was used to detect Influenza (A, B), metapneumovirus, respiratory syncytial virus (RSV), parainfluenza (1–4), rhinovirus, adenovirus (A—F), bocavirus and coronaviruses (NL63, 229E, OC43) in respiratory samples of children with acute respiratory infection prospectively admitted to any of the GENDRES network hospitals between 2011–2013. The results were corroborated in an independent cohort collected in the UK.

Results

A total of 204 and 97 nasopharyngeal samples were collected in the GENDRES and UK cohorts, respectively. In both cohorts, RSV was the most frequent pathogen (52.9% and 36.1% of the cohorts, respectively). Co-infection with multiple viruses was found in 92 samples (45.1%) and 29 samples (29.9%), respectively; this was most frequent in the 12–24 months age group. The most frequently observed co-infection patterns were RSV—Rhinovirus (23 patients, 11.3%, GENDRES cohort) and RSV—bocavirus / bocavirus—influenza (5 patients, 5.2%, UK cohort).

Conclusion

The presence of more than one virus in pediatric patients admitted to hospital with LT-ARI is very frequent and seems to peak at 12–24 months of age. The clinical significance of these findings is unclear but should warrant further analysis.     

Metabolic Abnormalities Are Common among South American Hispanics Subjects with Normal Weight or Excess Body Weight: The CRONICAS Cohort Study

Objective

We aimed to characterize metabolic status by body mass index (BMI) status.

Methods

The CRONICAS longitudinal study was performed in an age-and-sex stratified random sample of participants aged 35 years or older in four Peruvian settings: Lima (Peru’s capital, costal urban, highly urbanized), urban and rural Puno (both high-altitude), and Tumbes (costal semirural). Data from the baseline study, conducted in 2010, was used. Individuals were classified by BMI as normal weight (18.5–24.9 kg/m²), overweight (25.0–29.9 kg/m²), and obese (≥30 kg/m²), and as metabolically healthy (0–1 metabolic abnormality) or metabolically unhealthy (≥2 abnormalities). Abnormalities included individual components of the metabolic syndrome, high-sensitivity C-reactive protein, and insulin resistance.

Results

A total of 3088 (age 55.6±12.6 years, 51.3% females) had all measurements. Of these, 890 (28.8%), 1361 (44.1%) and 837 (27.1%) were normal weight, overweight and obese, respectively. Overall, 19.0% of normal weight in contrast to 54.9% of overweight and 77.7% of obese individuals had ≥3 risk factors (p<0.001). Among normal weight individuals, 43.1% were metabolically unhealthy, and age ≥65 years, female, and highest socioeconomic groups were more likely to have this pattern. In contrast, only 16.4% of overweight and 3.9% of obese individuals were metabolically healthy and, compared to Lima, the rural and urban sites in Puno were more likely to have a metabolically healthier profile.

Conclusions

Most Peruvians with overweight and obesity have additional risk factors for cardiovascular disease, as well as a majority of those with a healthy weight. Prevention programs aimed at individuals with a normal BMI, and those who are overweight and obese, are urgently needed, such as screening for elevated fasting cholesterol and glucose.     

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB₁ receptors peripherally modulate energy metabolism. Here, we investigated the role of CB₁ receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB₁ receptor antagonist AM251 (3 mg kg^-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB₁ receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB₁ receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB₁ receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB ₁ ^-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C₂C₁₂ myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB₁ receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.     

Diversity of Phytophthora Species from Declining Mediterranean Maquis Vegetation,including Two New Species,Phytophthora crassamura and P. ornamentata sp. nov.

The Mediterranean basin is recognized as a global biodiversity hotspot accounting for more than 25,000 plant species that represent almost 10% of the world’s vascular flora. In particular, the maquis vegetation on Mediterranean islands and archipelagos constitutes an important resource of the Mediterranean plant diversity due to its high rate of endemism. Since 2009, a severe and widespread dieback and mortality of Quercus ilex trees and several other plant species of the Mediterranean maquis has been observed in the National Park of La Maddalena archipelago (northeast Sardinia, Italy). Infected plants showed severe decline symptoms and a significant reduction of natural regeneration. First studies revealed the involvement of the highly invasive wide-host range pathogen Phytophthora cinnamomi and several fungal pathogens. Subsequent detailed research led to a better understanding of these epidemics showing that multiple Phytophthora spp. were involved, some of them unknown to science. In total, nine Phytophthora species were isolated from rhizosphere soil samples collected from around symptomatic trees and shrubs including Asparagus albus, Cistus sp., Juniperus phoenicea, J. oxycedrus, Pistacia lentiscus and Rhamnus alaternus. Based on morphological characters, growth-temperature relations and sequence analysis of the ITS and cox1 gene regions, the isolates were identified as Phytophthora asparagi, P. bilorbang, P. cinnamomi, P. cryptogea, P. gonapodyides, P. melonis, P. syringae and two new Clade 6 taxa which are here described as P. crassamura sp. nov. and P. ornamentata sp. nov. Pathogenicity tests supported their possible involvement in the severe decline that is currently threatening the Mediterranean maquis vegetation in the La Maddalena archipelago.     

Bacterial Factors Associated with Lethal Outcome of Enteropathogenic Escherichia coli Infection: Genomic Case-Control Studies

Background

Typical enteropathogenic Escherichia coli (tEPEC) strains were associated with mortality in the Global Enteric Multicenter Study (GEMS). Genetic differences in tEPEC strains could underlie some of the variability in clinical outcome.

Methods

We produced draft genome sequences of all available tEPEC strains from GEMS lethal infections (LIs) and of closely matched EPEC strains from GEMS subjects with non-lethal symptomatic infections (NSIs) and asymptomatic infections (AIs) to identify gene clusters (potential protein encoding sequences sharing ≥90% nucleotide sequence identity) associated with lethality.

Results

Among 14,412 gene clusters identified, the presence or absence of 392 was associated with clinical outcome. As expected, more gene clusters were associated with LI versus AI than LI versus NSI. The gene clusters more prevalent in strains from LI than those from NSI and AI included those encoding proteins involved in O-antigen biogenesis, while clusters encoding type 3 secretion effectors EspJ and OspB were among those more prevalent in strains from non-lethal infections. One gene cluster encoding a variant of an NleG ubiquitin ligase was associated with LI versus AI, while two other nleG clusters had the opposite association. Similar associations were found for two nleG gene clusters in an additional, larger sample of NSI and AI GEMS strains.

Conclusions

Particular genes are associated with lethal tEPEC infections. Further study of these factors holds potential to unravel the mechanisms underlying severe disease and to prevent adverse outcomes.     

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background

Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).

Methods/Principal Findings

The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.

Conclusions/Significance

Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.     

High Rate of Subclinical Chikungunya Virus Infection and Association of Neutralizing Antibody with Protection in a Prospective Cohort in The Philippines

Background

Chikungunya virus (CHIKV) is a globally re-emerging arbovirus for which previous studies have indicated the majority of infections result in symptomatic febrile illness. We sought to characterize the proportion of subclinical and symptomatic CHIKV infections in a prospective cohort study in a country with known CHIKV circulation.

Methods/Findings

A prospective longitudinal cohort of subjects ≥6 months old underwent community-based active surveillance for acute febrile illness in Cebu City, Philippines from 2012-13. Subjects with fever history were clinically evaluated at acute, 2, 5, and 8 day visits, and at a 3-week convalescent visit. Blood was collected at the acute and 3-week convalescent visits. Symptomatic CHIKV infections were identified by positive CHIKV PCR in acute blood samples and/or CHIKV IgM/IgG ELISA seroconversion in paired acute/convalescent samples. Enrollment and 12-month blood samples underwent plaque reduction neutralization test (PRNT) using CHIKV attenuated strain 181/clone25. Subclinical CHIKV infections were identified by ≥8-fold rise from a baseline enrollment PRNT titer <10 without symptomatic infection detected during the intervening surveillance period. Selected CHIKV PCR-positive samples underwent viral isolation and envelope protein-1 gene sequencing. Of 853 subjects who completed all study procedures at 12 months, 19 symptomatic infections (2.19 per 100 person-years) and 87 subclinical infections (10.03 per 100 person-years) occurred. The ratio of subclinical-to-symptomatic infections was 4.6:1 varying with age from 2:1 in 6 month-5 year olds to 12:1 in those >50 years old. Baseline CHIKV PRNT titer ≥10 was associated with 100% (95%CI: 46.1, 100.0) protection from symptomatic CHIKV infection. Phylogenetic analysis demonstrated Asian genotype closely related to strains from Asia and the Caribbean.

Conclusions

Subclinical infections accounted for a majority of total CHIKV infections. A positive baseline CHIKV PRNT titer was associated with protection from symptomatic CHIKV infection. These findings have implications for assessing disease burden, understanding virus transmission, and supporting vaccine development.     

Inhibition of Granulomatous Inflammation and Prophylactic Treatment of Schistosomiasis with a Combination of Edelfosine and Praziquantel

Background

Schistosomiasis is the third most devastating tropical disease worldwide caused by blood flukes of the genus Schistosoma. This parasitic disease is due to immunologic reactions to Schistosoma eggs trapped in tissues. Egg-released antigens stimulate tissue-destructive inflammatory and granulomatous reactions, involving different immune cell populations, including T cells and granulocytes. Granulomas lead to collagen fibers deposition and fibrosis, resulting in organ damage. Praziquantel (PZQ) is the drug of choice for treating all species of schistosomes. However, PZQ kills only adult Schistosoma worms, not immature stages. The inability of PZQ to abort early infection or prevent re-infection, and the lack of prophylactic effect prompt the need for novel drugs and strategies for the prevention of schistosomiasis.

Methodology/Principal Findings

Using in vitro and in vivo approaches, we have found that the alkylphospholipid analog edelfosine kills schistosomula, and displays anti-inflammatory activity. The combined treatment of PZQ and edelfosine during a few days before and after cercariae infection in a schistosomiasis mouse model, simulating a prophylactic treatment, led to seven major effects: a) killing of Schistosoma parasites at early and late development stages; b) reduction of hepatomegaly; c) granuloma size reduction; d) down-regulation of Th1, Th2 and Th17 responses at late post-infection times, thus inhibiting granuloma formation; e) upregulation of IL-10 at early post-infection times, thus potentiating anti-inflammatory actions; f) down-regulation of IL-10 at late post-infection times, thus favoring resistance to re-infection; g) reduction in the number of blood granulocytes in late post-infection times as compared to infected untreated animals.

Conclusions/Significance

Taken together, these data suggest that the combined treatment of PZQ and edelfosine promotes a high decrease in granuloma formation, as well as in the cellular immune response that underlies granuloma development, with changes in the cytokine patterns, and may provide a promising and effective strategy for a prophylactic treatment of schistosomiasis.     

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).