Advancement in the routine identification of anaerobic bacteria by MALDI-TOF mass spectrometry.

We evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) Biotyper as a tool for the identification of anaerobic bacteria compared with 500 base-pair (bp) 16S ribosomal ribonucleic acid (rRNA) gene sequencing analysis, which is considered to be the "gold standard" method. A total of 484 anaerobic bacteria were retrieved from the clinical specimens of 318 pediatric patients. Molecular identification resulted in 18 genera and 51 species. The most prevalent genus was Clostridium (76.85 %), with 70 % C. difficile isolates. The concordance and sensitivity determined by MALDI-TOF MS for C. difficile, the most prevalent species isolated, was 94.08 %, whereas the specificity was 100 %. For the other anaerobes, the sensitivity and specificity were 94.07 % and 81.82 %, respectively, with a concordance of 93.15 %. Low performance was observed for Propionibacterium acnes and Fusobacterium nucleatum, for which a dedicated pretreatment procedure should likely be set up. MALDI-TOF MS was shown to be a valid alternative for the fast and reliable identification of the most clinically relevant anaerobic bacteria; moreover, it is less time-consuming, the cost for reagents is minimized, and it does not require dedicated personnel.

Coinfection in acute gastroenteritis predicts a more severe clinical course in children.

The objectives of this study were to determine the incidence of enteric pathogens causing acute gastroenteritis (AGE) among hospitalized children in a large Italian hospital, to measure the incidence of coinfections, and to compare the clinical characteristics of those infected with one versus multiple agents. A prospective study was conducted from March 2010 to April 2011 at the Bambino Gesù Pediatric Hospital in Rome, Italy. All patients between 1 month and 16 years of age admitted to the Pediatric Department with a diagnosis of AGE were eligible for enrollment. Two stool samples for each patient were tested for gastrointestinal pathogens. We summarized the clinical severity of episodes, describing the duration of diarrhea, duration and frequency of vomiting, fever, and severity of dehydration. All the patients underwent medical evaluation with estimation of dehydration. One or more etiological agents were detected in 151 out of 232 patients (65.1%), while we did not detect any etiological agent in 81 (34.9%). Rotavirus was detected in 96 (63.6%), adenovirus in 17 (11.2%), norovirus in 7 (4.6%), toxin-producing Clostridium difficile in 23 (15.2%), Salmonella spp. in 15 (9.9%, B group in 12/15 and D group in 3/15), C. perfringens in 12 (7.9%), Campylobacter spp. in 6 (4%), and verotoxigenic Escherichia coli (VTEC) in 2 (1.3%). In 27 children out of 151 (17.9%), we found evidence of coinfection. Coinfection with rotavirus and toxin-producing C. difficile was the most common (63%). Children with coinfection had a more severe clinical presentation and had a higher probability to be severely dehydrated, independently of age and living community type.

Cases of cryptosporidiosis co-infections in AIDS patients: a correlation between clinical presentation and GP60 subgenotype lineages from aged formalin-fixed stool samples.

Nine cases of cryptosporidiosis co-infections in AIDS patients were clinically categorised into severe (patients 1, 3, 8 and 9), moderate (patients 4 and 5) and mild (patients 2, 6 and 7). Formalin-fixed faecal specimens from these patients were treated to obtain high quality DNA competent for amplification and sequencing of the 60-kDa glycoprotein (GP60) gene. Sequence analysis revealed that one patient was infected with Cryptosporidium hominis whereas the remaining eight patients were infected with C. parvum. Interestingly, the patients showing severe cryptosporidiosis harboured two subtypes within the C. parvum allelic family IIc (IIcA5G3 and IIcA5G3R2), whereas patients with moderate or mild infections showed various subtypes of the C. parvum allelic family IIa (IIaA14G2R1, IIaA15G2R1, IIaA17G3R1 and IIaA18G3R1). DNA extraction and genotyping of Cryptosporidium spp. is a challenging task on formalin-fixed stool samples, whose diagnostic outcome is age-dependent. The method herein reported represents a step forward routine diagnosis and improves epidemiology of HIV-related clinical cases. Due to the need to elucidate genetic richness of Cryptosporidium human isolates, this approach represents a useful tool to correlate individual differences in symptoms to subgenotyping lineages.

Investigation of Toxoplasma gondii presence in farmed shellfish by nested-PCR and real-time PCR fluorescent amplicon generation assay (FLAG).

To evaluate the presence of Toxoplasma gondii in edible farmed shellfish, 1734 shellfish specimens i.e., 109 Crassostrea gigas (6 pools), 660 Mytilus galloprovincialis (22 pools), 804 Tapes decussatus (28 pools) and 161 Tapes philippinarum (6 pools), were collected from the Varano Lagoon (Apulia, Italy). Shellfish from 62 pools were subjected to two molecular techniques: a nested-PCR assay, and a fluorescent amplicon generation (FLAG) real-time PCR assay, both based on the multi-copy B1 target, were performed. One pooled sample of gills from C. gigas and one pooled sample of haemolymphs from T. decussatus were assessed as positive for T. gondii DNA by both techniques. The results demonstrated the presence of T. gondii in edible farmed C. gigas and T. decussatus and indicate that there may be a considerable health threat involved in eating contaminated raw shellfish.

Human herpesvirus-8 serology in pediatric organ transplantation.

Human herpesvirus (HHV)-8 has been demonstrated to be involved in the pathogenesis of Kaposi's sarcoma, body cavity lymphomas, multicentric Castleman disease, and, more recently, acute bone marrow failure. In order to study the correlation between HHV-8 and immunosuppression, we performed a serological study in a group of 28 pediatric heart and heart-lung transplant recipients; of mean age 11.5 +/- 2.5 years. We analyzed blood samples prior to, at 3 to 6 months, and at least 12 months after transplantation. All patients were negative pretransplantation. We observed 10 seroconversions: one patient at 6 months; two patients at 12 months; and seven within the third year after transplantation. Our preliminary data demonstrated that HHV-8 seroconversion is frequent among pediatric transplant recipients. It was probably related to the length of immunosuppression rather than the organ transplantation; Serological assays of all donor specimens of seroconverted patients were negative.

Genotyping of different Pseudomonas aeruginosa morphotypes arising from the lower respiratory tract of a patient taken to an Intensive Care Unit.

Pseudomonas aeruginosa is an opportunistic pathogen and an ubiquitous environmental bacterium. Fifty-seven days after hospitalization, we isolated three distinct P. aeruginosa morphotypes (smooth, rough and mucoid) from the lower respiratory tract of a patient admitted to a Cardiology Intensive Care Unit (ICU). Moreover, a group of nine colony variants, arising from the three P. aeruginosa isolates growing in laboratory growth media, were also isolated. The resulting 12 isolates were characterised for antibiotic resistance profile and subjected to genotypic analysis by fluorescent-Amplified Fragment Length Polymorphism (f-AFLP) and automated repetitive extragenic palindromic-PCR (rep-PCR) fingerprinting. The three smooth, rough and mucoid morphotypes presented different antibiotic resistance profiles and genotyping analysis showed that they belonged to distinct clones, indicating that at day 57 after the admission the patient was simultaneously colonized by three distinct P. aeruginosa isolates. On the other hand, the nine colony variants presented heterogeneous antibiotic resistance profiles and clustered together with the three parental isolates. The understanding of the link between genotype plasticity and antibiotic resistance may contribute to improving our knowledge of this life-threatening pathogen.

[Role of screening tests for indirect diagnosis of tuberculosis in health care workers: Mantoux and the new tests on blood ELISA]. / Ruolo dei test di screening nella diagnosi indiretta di tubercolosi negli operatori sanitari: la Mantoux e il nuovo test ELISA su sangue.

The Tuberculosis infection in recent years has become always more a threat. The failure in the attempt to stop it (O.M.S. Millennium Global Plan) brought to the revision of the world control strategy to at least contain this disease (The Global Plan to Stop TB 2006-2015). Due to these severe facts it is even more important now to elaborate more sensitive and specific methods to find out, as fast as possible, the infected cases. As of today, the main TB infection screening test is the Skin PPD test (Mantoux). Recently new tests for the population screening are in use; these tests are based on the evaluation of immunity cell-mediated. They (QFT-G) do not have the typical limits of the Skin Test and they are more suitable as serial tests and therefore more useful, according to us, in the screening programs of the TB infection in low prevalence countries, like Italy.

Protein zero is necessary for E-cadherin-mediated adherens junction formation in Schwann cells.

Protein Zero (P0), the major structural protein in the peripheral nervous system (PNS) myelin, acts as a homotypic adhesion molecule and is thought to mediate compaction of adjacent wraps of myelin membrane. E-Cadherin, a calcium-dependent adhesion molecule, is also expressed in myelinating Schwann cells in the PNS and is involved in forming adherens junctions between adjacent loops of membrane at the paranode. To determine the relationship, if any, between P0-mediated and cadherin-mediated adhesion during myelination, we investigated the expression of E-cadherin and its binding partner, beta-catenin, in sciatic nerve of mice lacking P0 (P0(-/-)). We find that in P0(-/-) peripheral myelin neither E-cadherin nor beta-catenin are localized to paranodes, but are instead found in small puncta throughout the Schwann cell. In addition, only occasional, often rudimentary, adherens junctions are formed. Analysis of E-cadherin and beta-catenin expression during nerve development demonstrates that E-cadherin and beta-catenin are localized to the paranodal region after the onset of myelin compaction. Interestingly, axoglial junction formation is normal in P0(-/-) nerve. Taken together, these data demonstrate that P0 is necessary for the formation of adherens junctions but not axoglial junctions in myelinating Schwann cells.

Charcot-Marie-Tooth disease type 1: molecular pathogenesis to gene therapy.

Charcot-Marie-Tooth disease type 1 (CMT1) is caused by mutations in the peripheral myelin protein, 22 kDa (PMP22) gene, protein zero (P0) gene, early growth response gene 2 (EGR-2) and connexin-32 gene, which are expressed in Schwann cells, the myelinating cells of the peripheral nervous system. Although the clinical and pathological phenotypes of the various forms of CMT1 are similar, including distal muscle weakness and sensory loss, their molecular pathogenesis is likely to be quite distinct. In addition, while demyelination is the hallmark of CMT1, the clinical signs and symptoms of the disease are probably produced by axonal degeneration, not demyelination itself. In this review we discuss the molecular pathogenesis of CMT1, as well as approaches to an effective gene therapy for this disease.

Regulation of myelin-specific gene expression. Relevance to CMT1.

Schwann cells, the myelinating cells of the peripheral nervous system, are derived from the neural crest. Once neural crest cells are committed to the Schwann cell fate, they can take on one of two phenotypes to become myelinating or nonmyelinating Schwann cells, a decision that is determined by interactions with axons. The critical step in the differentiation of myelinating Schwann cells is the establishment of a one-to-one relationship with axons, the so-called "promyelinating" stage of Schwann cell development. The transition from the promyelinating to the myelinating stage of development is then accompanied by a number of significant changes in the pattern of gene expression, including the activation of a set of genes encoding myelin structural proteins and lipid biosynthetic enzymes, and the inactivation of a set of genes expressed only in immature or nonmyelinating Schwann cells. These changes are regulated mainly at the transcriptional level and also require continuous interaction between Schwann cells and their axons. Two transcription factors, Krox 20 (EGR2) and Oct 6 (SCIP/Tst1), are necessary for the transition from the promyelinating to the myelinating stage of Schwann cell development. Krox 20, expressed in myelinating but not promyelinating Schwann cells, is absolutely required for this transition, and myelination cannot occur in its absence. Oct 6, expressed mainly in promyelinating Schwann cells and then down-regulated before myelination, is necessary for the correct timing of this transition, since myelination is delayed in its absence. Neither Krox 20 nor Oct 6, however, is required for the initial activation of myelin gene expression. Although the mechanisms of Krox 20 and Oct 6 action during myelination are not known, mutation in Krox 20 has been shown to cause CMT1, further implicating this protein in the pathogenesis of this disease. Identifying the molecular mechanisms of Krox 20 and Oct 6 action will thus be important both for understanding myelination and for designing future treatments for CMT1. Point mutlations in the genes encoding the myelin proteins PMP22 and P0 cause CMT1A without a gene duplication and CMT1B, respectively. Although the clinical and pathological phenotypes of CMT1A and CMT1B are similar, their molecular pathogenesis is quite different. Point mutations in PMP22 alter the trafficking of the protein, so that it accumulates in the endoplasmic reticulum (ER) and intermediate compartment (IC). Mutant PMP22 also sequesters its normal counterpart in the ER, further reducing the amount of PMP22 available for myelin synthesis at the membrane, and accounting, at least in part, for its severe effect on myelination. Mutant PMP22 probably also activates an ER-to-nucleus signal transduction pathway associated with misfolded proteins, which may account for the decrease of myelin gene expression in Schwann cells in Trembler mutant mice. In contrast, absence of expression of the homotypic adhesion molecule, P0, in mice in which the gene has been inactivated, produces a unique pattern of Schwann cell gene expression, demonstrating that P0 plays a regulatory as well as a structural role in myelination. Whether this role is direct, through a P0-mediated adhesion pathway, or indirect, through adhesion pathways mediated by cadherins or integrins, however, remains to be determined. The molecular mechanisms underlying dysmyelination in CMT1 are thus complex, with pleitropic effects on Schwann cell physiology that are determined both by the type of mutation and the protein mutated. Identifying these molecular mechanisms, however, are important both for understanding myelination and for designing future treatments for CMT1. Although demyelination is the hallmark of CMT1, the clinical signs and symptoms of this disease are probably produced by axonal degeneration, not demyelination. (ABSTRACT TRUNCATED)

The absence of myelin P0 protein produces a novel molecular phenotype in Schwann cells.

In order to better understand the pathogenesis of demyelination in P0 knockout (P0-/-) mice, we analyzed the myelin gene expression and the localization of myelin proteins in P0 null mouse sciatic nerve. We have demonstrated that the severe demyelinating neuropathy of P0-knockout mouse is associated with changes in the program of myelin gene expression. Some changes in myelin gene expression occur early, others occur during adulthood. We also provide evidence that the absence of P0 is associated with changes in the localization of specific paranodal proteins in the peripheral nerve. These data suggest that P0 plays an important role, either directly or indirectly, in the program of Schwann cell gene expression and in the specific distribution of peripheral myelin proteins. Furthermore, myelin gene dysregulation and improper localization of paranodal proteins may account, in part, for the pathogenesis of demyelination in P0-knockout mice, as well as in human demyelinating peripheral neuropathy associated with mutations in the P0 gene.

Overcoming cellular immunity to prolong adenoviral-mediated gene expression in sciatic nerve.

In a previous report, we demonstrated that a first generation (E1- and E3-deleted) recombinant adenovirus can transduce expression of the E. coli lacZ gene into Schwann cells, both in vitro and in vivo, suggesting that this method might be useful for future therapy of peripheral neuropathy, including CMT1. Adenoviral-mediated gene transfer was limited, however, by demyelination and Wallerian degeneration at the site of virus injection, as well as by attenuation of viral gene expression over time. In our current work we have optimized adenoviral-mediated gene expression after intraneural injection into sciatic nerve. Using an improved injection protocol, peak expression of lacZ occurs between 10 and 14 days after injection of 2-week-old animals, decreases thereafter, and there is minimal associated tissue injury. In contrast, very few adenoviral-infected Schwann cells are found in nerves of adult animals 10 days after injection, probably due to immune clearance of viral-infected cells. Consistent with this notion, high levels of lacZ are found in sciatic nerve 30 days after injection of adult SCOD mice, which have a genetic defect in both cellular and humoral immunity, of adult beta 2 microglobulin-deficient mice (beta 2 M-/-), which have a genetic defect in cellular immunity, or of adult mice treated with the immunosuppressing agent FK506. In addition, adenoviral-infected Schwann cells co-cultured with axons in vitro, in the absence of a host immune response, ensheath axons and express lacZ for at least 8 weeks. These data thus demonstrate that expression of first generation recombinant adenovirus in sciatic nerve in adult mice, as in other tissues, is limited mainly by the host cellular immune response to the virus, which can be overcome by attenuation of host cell-mediated immunity. Adenoviral vectors might thus be used to modulate Schwann cell gene expression in patients with peripheral neuropathy after appropriate immunosuppression.

Modulation of cell-mediated immunity prolongs adenovirus-mediated transgene expression in sciatic nerve.

In a previous report, we demonstrated that a first-generation (E1- and E3-deleted) recombinant adenovirus can transduce expression of the E. coli lacZ gene into Schwann cells, both in vitro and in vivo, suggesting that this method might be useful for future therapy of peripheral neuropathy, including CMT1. Adenovirus-mediated gene transfer was limited, however, by demyelination and Wallerian degeneration at the site of virus injection, as well as by attenuation of viral transgene expression over time. In our current work we have optimized adenoviral vector-mediated transgene expression after intraneural injection into sciatic nerve. Using an improved injection protocol, peak expression of lacZ occurs between 10 and 14 days after injection of 2-week-old rats, decreases thereafter, and there is minimal associated tissue injury. In contrast, few lacZ-expressing Schwann cells are found in nerve of adult animals 10 days after injection, probably owing to immune clearance of virus-infected cells. Consistent with this notion, high levels of LacZ are found in sciatic nerve 30 days after injection of adult SCID mice, which have a genetic defect in both cellular and humoral immunity, of adult beta2-microglobulin-deficient mice (beta2M4-/-), which have a genetic defect in cellular immunity, or of adult mice treated with the immunosuppressing agent FK506. In addition, adenovirus-infected Schwann cells cocultured with axons in vitro, in the absence of a host immune response, ensheathe axons and express lacZ for at least 8 weeks. These data thus demonstrate that lacZ transgene expression of first-generation recombinant adenovirus in sciatic nerve in adult mice, as in other tissues, is limited mainly by the host cellular immune response to the virus, which can be overcome by attenuation of host cell-mediated immunity. Adenoviral vectors might thus be used to modulate Schwann cell gene expression in patients with peripheral neuropathy after appropriate immunosuppression.

Heterozygous P0 knockout mice develop a peripheral neuropathy that resembles chronic inflammatory demyelinating polyneuropathy (CIDP).

Demyelinating peripheral neuropathies are clinically divided into inherited and acquired types. Inherited demyelinating neuropathies are caused by mutations in genes expressed by myelinating Schwann cells, whereas acquired ones, including chronic inflammatory demyelinating polyneuropathy (CIDP), are probably caused by autoimmune mechanisms. We find that heterozygous P0 knockout (P0+/-) mice develop a neuropathy that resembles CIDP. By one year of age, P0+/- mice develop severe, asymmetric slowing of motor nerves, with temporal dispersion or conduction block, which are features of acquired demyelinating neuropathies including CIDP. Moreover, morphological analysis of affected nerves reveals severe and selective demyelination of motor fibers, focal regions of demyelination, and inflammatory cells. These data suggest that immune-mediated mechanisms may contribute to the pathogenesis of the neuropathy in P0+/- mice.

HHV-6 infection in Italy: characterization of an endemic isolate and seroepidemiologic analysis.

A biologic, immunologic and molecular characterization of an HHV-6 isolate (BA92) rescued by the peripheral blood mononuclear cells of a child affected by Exanthem subitum is reported. The comparison with the known HHV-6 prototype strains showed that BA92 is indistinguishable from the Z29 isolate, and can be included in the variant B group of HHV-6. A seroepidemiologic analysis of the antibody response to BA92 of normal individuals as well as patients affected by diseases potentially associated to HHV-6 infection has shown an overall seroprevalence of 81%, and that no variations in seroprevalence or in antibody geometric mean titer are observed assaying the sera also against G.S., U1102, or Z29 infected cells, respectively. These findings indicate: (1) HHV-6 infection is widely diffuse in Italy; (2) it is not possible to discriminate between the viral variants by the currently available IF assays, and (3) no conclusions can be drawn on the potential association of HHV-6 with any of the diseases examined.

Viral childhood diarrhoea in Rome: a diagnostic and epidemiological study.

During the period May 1987-January 1989, faecal samples from 417 paediatric inpatients admitted to the main paediatric hospital in Rome were screened by direct electron microscopy and rotavirus enzyme-linked immunosorbent assay. Rotaviruses were detected in 18.2% of cases and adenoviruses in 7%, whereas astroviruses were found in 1% of cases. Different percentages of rotavirus excretors were revealed by enzyme-linked immunosorbent assay and electron microscopy. This discrepancy seems to be due to false positive results introduced by enzyme-linked immunosorbent assay. Analysis of electron microscopy-positive samples by rotaviral RNA polyacrylamide gel electrophoresis showed different electropherotypes of rotavirus among which a single, largely predominant long electropherotype (55.4%) was revealed. Short electropherotype subgroup I rotaviruses were demonstrated in about 10.7% of samples.

Lymphocyte proliferative response to rabies virus antigen.

Rabies virus (RV) infection, as well as active immunisation using viral antigen, elicit both humoral and cellular reactions whose protective effects are still unclear. We evaluated both responses in order to find valuable monitoring parameters for the immunisation procedure. Three laboratory workers repetitively immunised with booster human diploid cell vaccine against rabies virus, 13 patients from the anti-rabies centre (vaccinated for the first time) and 10 healthy volunteers (not immunised nor exposed to rabies virus antigen), were monitored for: (i) in vivo RV-specific antibody production; (ii) in vitro anti-RV lymphocyte proliferative response and (iii) in vitro phenotype modulation induced by the viral antigen. In particular CD3, CD4, CD8, and surface immunoglobulins were monitored. All 3 subjects receiving the booster immunisation and, to a lesser extent, those receiving 4 doses of vaccine did recognise the antigen in vitro. The proliferation involved mainly CD4 positive cells leading to an increased number of cell bearing surface immunoglobulins, i.e. B cells. The proliferation index was in good correlation with the in vivo antibody production (p = less than 0.00009441). Nevertheless the presence of some cases without correlation between those parameters (in particular 5 out of 6 patients over 65 years of age failed to mount an adequate cellular proliferative response) reveals the need to use cellular response in parallel to the current humoral response, in order to evaluate and monitor the immunisation procedure. This conclusion is further stressed by the fact that protection against rabies infection is mainly cellular.

[Chlamydia trachomatis pneumonia in childhood. An underestimated problem?]. / Polmoniti da chlamydia trachomatis in età pediatrica. Un problema sottostimato?

The study of infections due to Chlamydia trachomatis in children has advanced remarkably during the past ten years. It has been established that Chlamydia trachomatis is a major etiologic agent of conjunctivitis in newborns and genital infections among sexually active persons. Chlamydial pneumonia has come to be recognized as one of the most common forms of pneumonia during the first three months of life. The Authors reported a case of a child admitted to Bambino Gesù Hospital of Rome, affected by Chlamydia trachomatis afebrile pneumonia. The diagnosis was made on the basis of direct and indirect laboratory findings. The remarkable improvement in direct and indirect diagnostic techniques can help the clinicians in diagnostic and therapeutic evaluations.