Clinical features of children hospitalized with influenza A and B infections during the 2012-2013 influenza season in Italy.

BACKGROUND: Influenza is a major public health issue worldwide. It is characterized by episodes of infection that involve hundreds of millions of people each year. Since that in the seasons 2010-2011 and 2011-2012 the circulation of FLUB was decreasing we evaluated the clinical presentation, demographic characteristics, admitting department, and length of stay in children who contracted influenza admitted to Bambino Gesù Children's Hospital, during the 2012-2013 influenza season, with the aim to establish if the recover of FLUB was associated to a clinical worsening, in comparison with those due to FLUA. METHODS: A total of 133 respiratory specimens, collected from patients with symptoms of respiratory tract infections, positive for the Influenza A and B viruses (FLUA and B) were subtyped. Comparisons between the FLUA and FLUB groups were performed with the one-way ANOVA for continuous parametric variables, the Mann-Whitney test for non-parametric variables, or the Chi-Square test or Fisher's exact test (if cells <5) for categorical variables. RESULTS: 87.09% of the FLUA isolates were the H1N1 subtype and 12.90% were H3N2. Among the FLUB isolates, 91.54% were the B/Yamagata/16/88 lineage and 8.45% were the B/Victoria/02/87 lineage. The largest number of FLUA/H1N1 cases was observed in children less than 1 years old, while the B/Yamagata/16/88 lineage was most prevalent in children 3-6 years old. Fever was a common symptom for both FLUA and B affected patients. However, respiratory symptoms were more prevalent in patients affected by FLUA. The median length of stay in the hospital was 5 days for FLUA and 3 days for FLUB. CONCLUSIONS: The clinical features correlated to different Influenza viruses, and relevant subtypes, were evaluated concluding that the increasing of FLUB in the season 2012-2013 was without any dramatic change in clinical manifestation. Our findings suggest, finally, that a stronger commitment to managing patients affected by FLUA is required, as the disease is more severe than FLUB.

Multiplex PCR allows rapid and accurate diagnosis of bloodstream infections in newborns and children with suspected sepsis.

Sepsis is a major health problem in newborns and children. Early detection of pathogens allows initiation of appropriate antimicrobial therapy that strongly correlates with positive outcomes. Multiplex PCR has the potential to rapidly identify bloodstream infections, compensating for the loss of blood culture sensitivity. In an Italian pediatric hospital, multiplex PCR (the LightCycler SeptiFast test) was compared to routine blood culture with 1,673 samples obtained from 803 children with suspected sepsis; clinical and laboratory information was used to determine the patient infection status. Excluding results attributable to contaminants, SeptiFast showed a sensitivity of 85.0% (95% confidence interval [CI] = 78.7 to 89.7%) and a specificity of 93.5% (95% CI = 92.1 to 94.7%) compared to blood culture. The rate of positive results was significantly higher with SeptiFast (14.6%) than blood culture (10.3%) (P < 0.0001), and the overall positivity rate was 16.1% when the results of both tests were combined. Staphylococcus aureus (11.6%), coagulase-negative staphylococci (CoNS) (29.6%), Pseudomonas aeruginosa (16.5%), and Klebsiella spp. (10.1%) were the most frequently detected. SeptiFast identified 97 additional isolates that blood culture failed to detect (24.7% P. aeruginosa, 23.7% CoNS, 14.4% Klebsiella spp., 14.4% Candida spp.). Among specimens taken from patients receiving antibiotic therapy, we also observed a significantly higher rate of positivity of SeptiFast than blood culture (14.1% versus 6.5%, respectively; P < 0.0001). On the contrary, contaminants were significantly more frequent among blood cultures than SeptiFast (n = 97 [5.8%] versus n = 26 [1.6%]), respectively; P < 0.0001). SeptiFast served as a highly valuable adjunct to conventional blood culture in children, adding diagnostic value and shortening the time to result (TTR) to 6 h.

Evaluation of a multiplex PCR assay for simultaneous detection of bacterial and viral enteropathogens in stool samples of paediatric patients.

We evaluated a multiplex PCR assay, the Seeplex Diarrhoea ACE detection, that simultaneously detects 15 enteric pathogens, including Salmonella spp., Shigella spp., Vibrio spp., toxin B producer Clostridium difficile, Campylobacter spp., Clostridium perfringens, Yersinia enterocolitica, Aeromonas spp., Escherichia coli O157:H7, verocytotoxin-producing Escherichia coli, adenovirus, Group A rotavirus, norovirus GI and GII, and astrovirus. We compared this assay with clinical methods routinely used in our laboratory, for detecting enteropathogens in stool samples collected from 245 paediatric patients with suspected infectious gastroenteritis. We recovered 61 bacterial pathogens and 121 enteric viruses with our laboratory assays, while we detected 78 bacteria and 167 viruses with the molecular assay. We calculated specificity and sensitivity for both methods after analysis of discordant results and demonstrated greater sensitivity for multiplex PCR than for our routine methods, with the exception of Salmonella spp. and toxigenic C. difficile detection. The multiplex PCR assay proved to be a reliable tool to directly detect the most common enteropathogens in stool samples but with some limitations.

MRSA nasal colonization in children: prevalence meta-analysis, review of risk factors and molecular genetics.

BACKGROUND: We report a meta-analysis of methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization prevalence in children and a review of the risk factors as well as molecular genetic characterization. METHODS: All relevant studies reporting prevalence data on MRSA nasal colonization in children published between January 2000 and August 2010 were retrieved from the MEDLINE database and analyzed. RESULTS: After screening 544 studies, 50 studies were included. We obtained an estimate of MRSA prevalence of 2.7% (95% confidence interval [CI]: 2.2-3.1); of 5.2% (95% CI: 3.1-7.3) in children with underlying conditions and of 2.3% (95% CI: 1.8-2.7) in healthy children; 5.4% (95% CI: 3.1-7.7) in children recruited in hospitals and 3% (95% CI: 2.4-3.6) in children recruited in the community. Staphylococcal cassette chromosome mec type IV is the most diffused cassette globally. CONCLUSION: The hospital remains the environment where the microorganism circulates most. Children with underlying conditions could act as vectors of microorganisms between the hospital and the community. MRSA prevention strategies should be tailored to each specific institution, taking into account the nosocomial prevalence of MRSA nasal colonization and infections, and the prevalence of nasal colonization in the community that refers to the specific health care center.

MALDI-TOF mass spectrometry proteomic phenotyping of clinically relevant fungi.

Proteomics is particularly suitable for characterising human pathogens with high life cycle complexity, such as fungi. Protein content and expression levels may be affected by growth states and life cycle morphs and correlate to species and strain variation. Identification and typing of fungi by conventional methods are often difficult, time-consuming and frequently, for unusual species, inconclusive. Proteomic phenotypes from MALDI-TOF MS were employed as analytical and typing expression profiling of yeast, yeast-like species and strain variants in order to achieve a microbial proteomics population study. Spectra from 303 clinical isolates were generated and processed by standard pattern matching with a MALDI-TOF Biotyper (MT). Identifications (IDs) were compared to a reference biochemical-based system (Vitek-2) and, when discordant, MT IDs were verified with genotyping IDs, obtained by sequencing the 25-28S rRNA hypervariable D2 region. Spectra were converted into virtual gel-like formats, and hierarchical clustering analysis was performed for 274 Candida profiles to investigate species and strain typing correlation. MT provided 257/303 IDs consistent with Vitek-2 ones. However, amongst 26/303 discordant MT IDs, only 5 appeared "true". No MT identification was achieved for 20/303 isolates for incompleteness of database species variants. Candida spectra clustering agreed with identified species and topology of Candida albicans and Candida parapsilosis specific dendrograms. MT IDs show a high analytical performance and profiling heterogeneity which seems to complement or even outclass existing typing tools. This variability reflects the high biological complexity of yeasts and may be properly exploited to provide epidemiological tracing and infection dispersion patterns.

Global distribution, public health and clinical impact of the protozoan pathogen cryptosporidium.

Cryptosporidium spp. are coccidians, oocysts-forming apicomplexan protozoa, which complete their life cycle both in humans and animals, through zoonotic and anthroponotic transmission, causing cryptosporidiosis. The global burden of this disease is still underascertained, due to a conundrum transmission modality, only partially unveiled, and on a plethora of detection systems still inadequate or only partially applied for worldwide surveillance. In children, cryptosporidiosis encumber is even less recorded and often misidentified due to physiological reasons such as early-age unpaired immunological response. Furthermore, malnutrition in underdeveloped countries or clinical underestimation of protozoan etiology in developed countries contribute to the underestimation of the worldwide burden. Principal key indicators of the parasite distribution were associated to environmental (e.g., geographic and temporal clusters, etc.) and host determinants of the infection (e.g., age, immunological status, travels, community behaviours). The distribution was geographically mapped to provide an updated picture of the global parasite ecosystems. The present paper aims to provide, by a critical analysis of existing literature, a link between observational epidemiological records and new insights on public health, and diagnostic and clinical impact of cryptosporidiosis.

Quantitative recovery of proviral HIV-1 DNA from leukocytes by the Dried Buffy Coat Spot method for real-time PCR determination.

The current recommended method for diagnosing HIV-1 in newborns infected vertically and in adults, during the "window period", is the detection of proviral HIV-1 DNA within leukocytes (buffy coat). This study describes a new portable Dried Buffy Coat Spot (DBCS) assay able to provide a quantitative proviral HIV-1 DNA recovery from the buffy coat. Fifty blood samples were collected from HIV-positive children and processed for DBCSs. Total DNA and proviral DNA were normalised to ß-globin and HIV-1 pol genes. Assay sensitivity and specificity were evaluated against the whole blood dried blood spot (DBS) method. Both procedures, using automatic DNA extraction, were compared to a standard whole blood DNA manual extraction. DNA recovery from whole blood was nearly equivalent to that of the DBCS-based extraction, while DBS-based extraction was 10-fold less sensitive. The detection rate of proviral HIV-1 DNA with DBCS assay was equivalent to whole blood manual extraction (100% concordance), but DBS-extracted samples showed limited concordance (44%). The DBCS assay may prove to be more feasible in resource-limited settings. It may represent a simple and robust point-of-care assay for HIV screening of children, for whom a reference test is still lacking.

Evaluation of the new GenoType Mycobacterium assay for identification of mycobacterial species.

The new commercial assay GenoType Mycobacterium, intended for the identification of mycobacteria, was evaluated with a panel of 197 strains belonging to 86 different taxa. The system, which relies on solid-phase reverse hybridization, is composed of two different kits: GenoType CM for the identification of more frequently detected mycobacterial species and GenoType AS for less common species. The sensitivity and specificity were 97.9% and 92.4% for the CM kit and 99.3% and 99.4% for the AS kit. Our results show that the system may represent a useful tool to substantially enlarge the number of mycobacteria that can be reliably identified in clinical laboratories.