Laura Becciolini

Community-Acquired Bacteremic Pneumococcal Pneumonia in Children: Diagnosis and Serotyping by Real-Time Polymerase Chain Reaction Using Blood Samples

BACKGROUND The aim of this study was to use real-time polymerase chain reaction (RT-PCR) on blood samples to diagnose and serotype pneumococcal infection in a large cohort of Italian children hospitalized for community-acquired pneumonia. METHODS We conducted an observational study from April 2007 through June 2009 of children aged 0-16 years with a diagnosis of community-acquired pneumonia admitted to 83 pediatric hospitals in Italy. RESULTS Seven hundred fifty-three children were studied. RT-PCR found pneumococcal infection in 80 (10.6%) of 753 patients. In 292 patients, culture and RT-PCR were simultaneously performed. Streptococcus pneumoniae was identified in 47 of 292 patients; 45 (15.4%) tested positive by RT-PCR and 11 (3.8%) tested positive by culture. RT-PCR was significantly more sensitive than culture in revealing bacteremic pneumonia (odds ratio, 30.6; 95% confidence interval, 5.8-97.5; P<.001). Complicated pneumonia was found in 162 (21.5%) of 753 children; 152 (93.8%) of these 162 had parapneumonic effusion, and 51 (33.6%) had empyema. Children with complicated pneumonia were significantly older. Pneumococcal bacteremia was found by RT-PCR to occur significantly more frequently in children with complications (38 [23.5%] of 162) than in children with uncomplicated pneumonia (44 [7.4%] of 591; odds ratio, 3.8; 95% confidence interval, 2.30-6.30; P<.001). RT-PCR allowed serotyping from blood in 92.5% of patients. More than two-thirds of the pneumonia cases were due to nonpneumococcal conjugate vaccine 7 serotypes. Serotype 1 was the most frequent serotype (26 [32.5%] of 80) and was significantly associated with complications (50.0% in patients with complicated pneumonia vs 18.2% in patients with uncomplicated pneumonia; odds ratio, 4.5, 95% confidence interval, 1.48-14.03; P=.005) and older age. Serotype 19A was second in frequency (15.0%) and was significantly associated with younger age. CONCLUSIONS RT-PCR allows diagnosis and serotyping of pneumococcal bacteremic community-acquired pneumonia in children and is an important tool for evaluating serotype distribution in culture-negative samples.

Interferon-Gamma Release Assay Improves the Diagnosis of Tuberculosis in Children

Background: Interferon-&ggr; release assays (IGRAs) have been recently developed for the diagnosis of tuberculosis (TB) infection. The aim of the present study was to evaluate the performance of an enzyme-linked immunosorbent assay (ELISA)-based IGRA for detecting TB in children. Methods: A prospective study in 336 children at risk for TB infection was carried out. All children were tested with tuberculin skin test (TST) and a commercial ELISA-based IGRA [QuantiFERON-TB Gold In-Tube (Cellestis)]. Results: TST were positive in 58 of 336 (17.3%) and IGRA in 60 of 336 (17.9%) children. Two (0.6%) IGRA results were indeterminate. The overall agreement between the 2 tests was intermediate (86.2%, κ = 0.533). IGRA was positive in 15 of 16 (93.8%) children with active pulmonary TB. The discordant pattern IGRA−/TST+ was significantly associated with Bacille Calmette-Guérin (BCG) vaccination. Among IGRA+ children (excluding cases of TB disease), TST− were significantly younger than TST+ children. Conclusions: The good agreement between positive IGRA and active TB disease suggests a good sensitivity of IGRA. Discrepancies between IGRA and TST can be a result of higher specificity of IGRA that is not influenced by previous BCG vaccination. IGRA may be more sensitive in children younger than 48 months.

Sphingosine 1-phosphate regulates myogenic differentiation: a major role for S1P2 receptor

In this study a novel biological activity of sphingosine 1‐phosphate (S1P) in C2C12 myoblasts was identified. In these cells the bioactive lipid profoundly regulated myogenesis exerting an antimitogenic activity, by reducing serum‐induced cell proliferation, and acting as powerful prodifferentiating agent by enhancing the expression of myogenic differentiation markers such as myogenin, myosin heavy chain, and caveolin‐3. The S1P‐dependent diminution of serum‐induced labeled thymidine incorporation was abrogated by antisense oligodeoxyribonucleotides (ODN) to S1P2, but not to S1P1 or S1P3 receptor, also expressed in C2C12 cells, implicating S1P2 in the biological response. Using antisense ODN and short interfering RNA treatment, we highlighted the key role played by S1P2 in the S1P‐dependent induction of muscle‐specific gene products. Notably, S1P2 overexpression increased the content of myogenic markers and hastened the onset of differentiated muscle phenotype in comparison with control cells. Cell treatment with pertussis toxin did not affect the biological responses to S1P, ruling out the involvement of Gi‐mediated events in the signaling promoted by the sphingolipid. Among the various signaling pathways activated by S1P, the activation of ERK1/ERK2 and p38 MAPK, both identified as downstream effectors of S1P2, was required for the inhibition of cell proliferation and the stimulation of myogenic differentiation, respectively.

Molecular detection methods and serotyping performed directly on clinical samples improve diagnostic sensitivity and reveal increased incidence of invasive disease by Streptococcus pneumoniae in Italian children

The aims of this study were to evaluate the incidence of invasive pneumococcal disease (IPD) in Italian children and perform serotyping by PCR-based assays directly on clinical samples. A 1-year paediatric (0–14 years) population-based surveillance study was designed to evaluate the incidence of IPD in the province of Florence, Italy, by cultural and molecular methods. Among 92 children (80 with pneumonia, 8 with meningitis/sepsis, 4 with arthritis), 4 cases of IPD were diagnosed both by culture and real-time PCR and 18 cases exclusively by molecular methods. The sensitivity of molecular methods was significantly higher than that of cultural methods (Cohen’s κ 0.41; McNemar P=0.000008). The incidence of IPD in children below 2 years of age was 11.5/100 000 and 51.8/100 000 by cultural and molecular methods, respectively. Pneumococcal serotyping by multiplex sequential PCR was obtained in 19/22 samples. Real-time PCR and multiplex sequential PCR can be used directly on biological samples, improving the ability to diagnose IPD. The incidence of IPD appears 5–10 times higher by PCR than by cultural methods.

Realtime PCR Is More Sensitive than Multiplex PCR for Diagnosis and Serotyping in Children with Culture Negative Pneumococcal Invasive Disease

Background Pneumococcal serotyping is usually performed by Quellung reaction, considered the gold standard test. However the method cannot be used on culture-negative samples. Molecular methods can be a useful alternative. The aim of the study was to evaluate the use of Multiplex-sequential-PCR (MS-PCR) or Realtime-PCR on blood samples for diagnosis and serotyping of invasive pneumococcal disease (IPD) in a pediatric clinical setting. Methodology/Principal Findings Sensitivity and specificity of MS-PCR and Realtime-PCR have been evaluated both on 46 well characterized pneumococcal isolates and on 67 clinical samples from children with culture-negative IPD. No difference in sensitivity and specificity between MS-PCR and Realtime PCR was found when the methods were used on isolates: both methods could type 100% isolates and the results were always consistent with culture-based methods. On the contrary, when used on clinical samples 43/67 (64.2%) were typeable by MS-PCR and 61/67 (91.0%) by Realtime-PCR (p = 0.0004,K Cohen 0.3, McNemars p<0.001). Non-typeability by MS-PCR was associated in 18/20 cases (90.0%) with low bacterial load. The difference between the two methods was present both when they were used on normally sterile fluids (respectively 31/33 (93.9%) typeable samples for Realtime-PCR and 24/33 (72.7%) for MS-PCR, p = 0.047, 95%CL 0.03–0.98; K Cohen 0.3; McNemars p = 0.0016) and when they were used on nasopharyngeal swabs (respectively 30/34 (88.2%) typeable samples for Realtime-PCR and 19/34 (55.9%) for MS-PCR, p = 0.007, 95%CL 0.04–0.66); the presence of multiple pneumococcal serotypes in nasopharyngeal swabs was found more frequently by Realtime PCR (19/30; 63.3%) than by Multiplex-sequential PCR (3/19; 15.8%; p = 0.003;95%CL 1.87–39.97). Conclusions/Significance In conclusion, both MS-PCR and Realtime PCR can be used for pneumococcal serotyping of most serotypes/serogroups directly on clinical samples from culture-negative patients but Realtime-PCR appears more sensitive.

Pneumococcal DNA is not detectable in the blood of healthy carrier children by real-time PCR targeting the lytA gene

The diagnosis of invasive pneumococcal disease (IPD) is currently based on culture methods, which lack sensitivity, especially after antibiotic therapy. Molecular methods have improved sensitivity and do not require viable bacteria; however, their use is complicated by reports of low specificity with some assays. The present study investigated the specificity of a real-time PCR targeting lytA for the detection of IPD. A group of 147 healthy children, aged 6 months to 16 years (mean 6.4 years, median 4.9 years, interquartile range 6.4 years), who were in hospital for routine examinations, were tested for pneumococcal carrier status and for the presence of detectable pneumococcal DNA in their blood by real-time PCR targeting the pneumococcal lytA gene. In addition, 35 culture-positive biological samples were analysed. Urine was examined for the presence of pneumococcal DNA and C-polysaccharide antigen. Carriage was detected in 77 of the 147 subjects (52.4 %); however, regardless of carrier status, none of the subjects had a positive result from blood. Analysis of the culture-positive biological samples yielded positive results in 100 % (15/15) of cerebrospinal fluid samples and 95 % (19/20) of blood samples. All urine samples from healthy carriers were negative for DNA, whilst antigenuria was detected in 44/77 carriers (57.1 %). In conclusion, real-time PCR is both sensitive and specific and can be a useful tool in the routine diagnosis of IPD. Its sensitivity, which surpasses that of other methods for this purpose, does not come at the cost of reduced specificity.

Comparison of the effect of antibiotic treatment on the possibility of diagnosing invasive pneumococcal disease by culture or molecular methods: A prospective, observational study of children and adolescents with proven pneumococcal infection

BACKGROUND Detection of Streptococcus pneumoniae in culture specimens in invasive pneumococcal disease (IPD) may be hampered by antibiotic treatment administered before hospital admission. Realtime polymerase chain reaction (RT-PCR) assays do not require viable bacteria and are therefore less influenced by antimicrobial therapy. It is not known how long results of culture or molecular tests remain positive after antibiotic therapy is begun. OBJECTIVE The goal of the current study was to assess, in a pediatric population with a diagnosis of IPD confirmed by laboratory tests (culture and/or RT-PCR assay), the relationship between use of antibiotic therapy before hospital admission and the result of diagnostic methods (culture or molecular techniques) after admission. METHODS This prospective, observational study was conducted from April 2006 through March 2009. All children and adolescents aged 0 to 16 years, admitted to the hospital with a diagnosis of IPD confirmed by culture and/or molecular methods, were included in the study. Previous antibiotic treatment (drug, duration of therapy) was recorded. Primers and probes designed from the pneumococcal autolysin gene (lytA) were used in an RT-PCR assay for detection of S pneumoniae. Antibiotic tolerability, permanent sequelae (after a 6-month follow-up), and deaths were recorded. RESULTS Eighty-three patients (50 males, 33 females; 80 white, 3 Asian; mean age, 4.6 years; median age, 4.0 years; age range, 10 days-16 years) were included in the study. Fifty-four patients presented with pneumonia, 26 with meningitis/sepsis (meningitis, 19; sepsis, 7), and 3 with arthritis. Results of RT-PCR assays were positive in all 83 patients (100.0%), and 28 of the 83 patients (33.7%) also had culture-positive findings. Forty-two of the 83 patients (50.6%) had received antibiotic treatment before hospital admission, and 41 (49.4%) had not received antibiotics. Results of cultures were positive in 9 of the 42 patients with IPD (21.4%) who had received antibiotic treatment and in 19 of the 41 patients with IPD (46.3%) who had not received antibiotics (odds ratio, 3.2; 95% CI, 1.1-9.3; P = 0.03). Molecular methods appeared more sensitive than culture in any type of disease studied but particularly in patients with pneumonia, in whom the difference was statistically significant (P = 0.043). The mean length of antibiotic therapy was 1.4 days (median, 1 day; SD, 0.53 day; range, 1-2 days) for culture-confirmed cases and 4.5 days (median, 4 days; SD, 3.08 days; range, 1-15 days) for cases confirmed by RT-PCR assay (P = 0.002). No adverse reactions to the antibiotics used during home or hospital treatment were found. Two patients with meningitis suffered permanent, severe neurologic sequelae, and 1 girl died of sepsis 3 days after hospital admission. No permanent sequelae were recorded in patients with pneumonia or arthritis. CONCLUSION In these children and adolescents with IPD, the molecular methods used appeared to be more sensitive than culture in any IPD patient, with a higher statistical significance in patients previously treated with antibiotics and in patients with pneumonia.

Localization of neutral ceramidase in caveolin-enriched light membranes of murine endothelial cells

Sphingomyelinase (SMase) and ceramidase (CDase) activities participate in sphingomyelin (SM) metabolism and have a role in the signal transduction of a variety of ligands. In this study evidence is presented that caveolin‐enriched light membranes (CELMs) of murine endothelial cells, characterized by high SM, ceramide (Cer) and cholesterol content, bear acid and neutral SMase as well as neutral CDase activities. Localization of neutral CDase in CELMs was confirmed by Western analysis. Notably, cell treatment with cyclodextrin, which depleted cell cholesterol, did not affect acid or neutral SMase activities but significantly enhanced neutral CDase activity in CELMs, indicating a negative role for cholesterol in CDase regulation. These findings suggest that neutral CDase is implicated, together with SMase activities, in the control of caveolar Cer content that may be critical for caveola dynamics.

Sphingosine kinase activity is required for sphingosine-mediated phospholipase D activation in C2C12 myoblasts

Sphingosine (Sph) has been implicated as a modulator of membrane signal transduction systems and as a regulatory element of cardiac and skeletal muscle physiology, but little information is presently available on its precise mechanism of action. Recent studies have shown that sphingosine 1-phosphate (S1P), generated by the action of sphingosine kinase (SphK) on Sph, also possesses biological activity, acting as an intracellular messenger, as well as an extracellular ligand for specific membrane receptors. At present, however, it is not clear whether the biological effects elicited by Sph are attributable to its conversion into S1P. In the present study, we show that Sph significantly stimulated phospholipase D (PLD) activity in mouse C2C12 myoblasts via a previously unrecognized mechanism that requires the conversion of Sph into S1P and its subsequent action as extracellular ligand. Indeed, Sph-induced activation of PLD was inhibited by N,N-dimethyl-D-erythro-sphingosine (DMS), at concentrations capable of specifically inhibiting SphK. Moreover, the crucial role of SphK-derived S1P in the activation of PLD by Sph was confirmed by the observed potentiated effect of Sph in myoblasts where SphK1 was overexpressed, and the attenuated response in cells transfected with the dominant negative form of SphK1. Notably, the measurement of S1P formation in vivo by employing labelled ATP revealed that cell-associated SphK activity in the extracellular compartment largely contributed to the transformation of Sph into S1P, with the amount of SphK released into the medium being negligible. It will be important to establish whether the mechanism of action identified in the present study is implicated in the multiple biological effects elicited by Sph in muscle cells.

Intrafamilial transmission of hepatitis C virus: Infection of the father predicts the risk of perinatal transmission

The aims of the present study were to evaluate in a cohort of mothers infected with hepatitis C virus (HCV) the prevalence of HCV infection of their sexual partners, the influence of infection of the partners on perinatal transmission, and whether this influence is mediated by other well known risk factors for perinatal transmission. Forty‐nine consecutive mothers infected with HCV who transmitted infection to their offspring and, as a control group, 557 consecutive mothers infected with HCV who did not transmit infection, together with their children and the fathers of the children who were also the sexual partners of the mothers were evaluated. History of intravenous drug use was significantly more frequent in women with partners infected with HCV than in women with partners not infected [115/180 (63.9%) vs. 87/401 (21.7%); relative risk (RR): 6.38, 95% confidence intervals (CI): 4.34–9.39, P < 10−3]. HCV infection was more frequent in the partners of mothers who transmitted perinatally HCV [23/49 (46.9%) vs. 174/557 (31.2%); RR: 1.95, 95%CI: 1.08–3.51, P = 0.03]. Multivariate analysis demonstrated that paternal HCV infection is not a risk factor per se for perinatal HCV transmission, but its role is dependent on maternal intravenous drug use [adjusted RR: 1.23 (95%CI: 0.44–3.39, P = 0.6)]. In conclusion, the present study shows that partners of mothers infected with HCV with a history of intravenous drug use were at a higher risk of HCV infection. HCV infection of the father seems to be associated with perinatal transmission but this relationship is dependent on maternal history of intravenous drug use. J. Med. Virol. 80:1907–1911, 2008.