Eddi Di Marco

Epidemiology and clinical features of Mycoplasma pneumoniae infection in children

Mycoplasma pneumoniae (MP) is, considered to affect rarely children less than 5 yrs of age. This study was performed to describe the epidemiology and the clinical features of MP lower respiratory tract infection (LRTI) in children, presenting to a tertiary children hospital. Eleven month-longitudinal study of LRTI due to MP, diagnosed by polymerase chain reaction (PCR) on throat swab specimen, was performed. Out of 866 children with LRTI admitted to the Gaslini Pediatric Institute in Genoa, 102 had a positive PCR for MP. We found 39 preschool-aged children, 42 school-aged children and 21 young adolescent [6.20 (3.81) yrs old]. Interestingly, eight MP+ infants had <8 months of age. The commonest presentations were cough and/or fever (76.5%). Tachypnoea, upper respiratory tract involvement, diarrhoea and vomiting were more common in the <5 yr Gr as compared to the other groups. Chest X-ray was found abnormal in 76 children: consolidations were the commonest finding. Laboratory test showed that the preschool-aged children had a higher number of lymphocytes (p<0.0001) and monocytes (p=0.009). Thrombocytosis was found in 35.7% of children and was more frequent in the preschool-aged children (p=0.013). MP infection is common in preschool-aged children, including young infants, and may have different clinical presentation, as compared to older children.

Low dosage cidofovir without probenecid as treatment for BK virus hamorrhagic cystitis after hemopoietic stem cell transplant.

We describe a single-center pediatric experience with 1 mg/kg/wk cidofovir without probenecid in 7 children with BK virus-associated hemorrhagic cystitis. Clinical improvement was observed in all cases, without adverse events, although significant reduction of urinary viral load was observed 2 weeks after the end of cidofovir in 5 out of 6 patients who completed the treatment.

HHV-8-related visceral Kaposi’s sarcoma following allogeneic HSCT: Report of a pediatric case and literature review

Sala I, Faraci M, Magnano GM, Sementa A, di Marco E, Garaventa A, Micalizzi C, Lanino E, Morreale G, Moroni C, Castagnola E. HHV‐8‐related visceral Kaposi’s sarcoma following allogeneic HSCT: Report of a pediatric case and literature review.
Pediatr Transplantation 2011: 15:E8–E11.

A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2

Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples.

The administration of a polyvalent mechanical bacterial lysate in elderly patients with COPD results in serological signs of an efficient immune response associated with a reduced number of acute episodes

The administration of a polyvalent mechanical bacterial lysate (PMBL) in elderly patients with COPD has been shown to reduce the number of exacerbation. This is largely related to the involvement of cells belonging to the innate and the adaptive immune system (including dendritic cells, granulocytes, T and B lymphocytes and NK cells) that actively cooperate inducing the production of specific opsonizing antibodies directed to the antigens of PMBL. We have evaluated the production of antibodies directed to respiratory and systemic pathogens in a group of elderly COPD patients, recruited in a clinical trial, ancillary to a larger multicenter double blind, placebo-controlled, parallel-designed clinical trial in which patients were randomized to daily receive either PMBL or placebo. The treated group not only experienced a reduced number of seroconversion, but also, better controlled the number of infectious episodes and COPD exacerbations. It was thus evident that the administration of PMBL resulted not only effective in inducing the secretion of specific antibodies, but also effective in reducing the infectious episodes trough the potentiation of the antibody-mediated arm of the immune response.

Activating Killer Immunoglobulin Receptors and HLA-C: A successful combination providing HIV-1 control

Several studies demonstrated a relevant role of polymorphisms located within the HLA-B and -C loci and the Killer Immunoglobulin Receptors (KIRs) 3DL1 and 3DS1 in controlling HIV-1 replication. KIRs are regulatory receptors expressed at the surface of NK and CD8+ T-cells that specifically bind HLA-A and -B alleles belonging to the Bw4 supratype and all the -C alleles expressing the C1 or C2 supratype. We here disclose a novel signature associated with the Elite Controller but not with the long-term nonprogressor status concerning 2DS activating KIRs and HLA-C2 alleles insensitive to miRNA148a regulation. Overall, our findings support a crucial role of NK cells in the control of HIV-1 viremia.

Real-Time PCR Detection of Mycoplasma pneumoniae in the Diagnosis of Community-Acquired Pneumonia

Polymerase chain reaction is a useful technique in microbial diagnostics to detect and quantify DNA or RNA of low abundance. Bacterial and viral nucleic acid can be amplified by PCR upon clinical sample extraction using specific primers for classical qualitative PCR and primers and probes for real-time PCR. Here we describe the Scorpion-probe real-time PCR-based assay that offers thermodynamic advantages due to its kinetic reaction and provides faster performances compared to a classical double-labeled probe-based assays.Polymerase chain reaction is a useful technique in microbial diagnostics to detect and quantify DNA or RNA of low abundance. Bacterial and viral nucleic acid can be amplified by PCR upon clinical sample extraction using specific primers for classical qualitative PCR and primers and probes for real-time PCR. Here we describe the Scorpion-probe real-time PCR-based assay that offers thermodynamic advantages due to its kinetic reaction and provides faster performances compared to a classical double-labeled probe-based assays.

The bacterial lysate Lantigen B reduces the number of acute episodes in patients with recurrent infections of the respiratory tract: the results of a double blind, placebo controlled, multicenter clinical trial.

Abstract Studies in the 1970s and 1980s reported that bacterial lysates (BL) had a prophylactic effect on recurrent respiratory tract infections (RRTI). However, controlled clinical study procedures have evolved substantially since then. We performed a trial using updated methods to evaluate the efficacy of Lantigen B®, a chemical BL. This double blind, placebo controlled, multi-center clinical trial had the primary objective of assessing the capacity of Lantigen B to significantly reduce the total number of infectious episodes in patients with RRTI. Secondary aims were the RRTI duration, the frequency and the severity of the acute episodes, the use of drugs and the number of missed workdays. In the subgroup of allergic patients with RRTI, the number of allergic episodes (AE) and the use of anti-allergic drugs were also evaluated. One hundred and sixty patients, 79 allocated to the treated group (TG) and 81 to the placebo group (PG), were enrolled; 30 were lost during the study and 120 (79 females and 38 males) were evaluated. The PG had 1.43 episodes in the 8-months of follow-up while the TG had 0.86 episodes (p =0.036). A similar result was observed in the allergic patients (1.80 and 0.86 episodes for the PG and the TG, respectively, p =0.047). The use of antibiotics was reduced (mean 1.24 and 2.83 days of treatment for the TG and the PG). Logistic regression analysis indicated that the estimated risk of needing antibiotics and NSAIDs was reduced by 52.1 and 30.6%, respectively. With regard to the number of AE, no significant difference was observed between the two groups, but bronchodilators, antihistamines and local corticosteroids were reduced by 25.7%, 56.2% and 41.6%, respectively, in the TG. Lantigen B significantly reduced the number of infectious episodes in patients with RRTI. This finding suggests a first line use of this drug for the prophylaxis of infectious episodes in these patients.

Pneumonia due to Mycoplasma pneumoniae in granulocytopenic children with cancer.

To the Editor: Mycoplasma pneumoniae causes communityacquired pneumonias in children and adults [1]. Diagnosis is based on clinical symptoms, radiological signs, and detection of IgM and IgG by enzyme-linked immunosorbent assay, but polymerase chain reaction (PCR) on a throat swab specimen represents a specific and rapid diagnostic method [1]. Oddly, M. pneumoniae is not included among the possible causes of pneumonia in immunocompromised hosts [2] and a comprehensive MEDLINE search yielded only a total of 11 cases [3–5]. After the observation of a severe pneumonia with diffuse bilateral lung infiltrates and hemolytic anemia due to M. pneumoniae in a splenectomized patient previously treated for Hodgkin disease, we checked for M. pneumoniae infection in pneumonias observed in children with cancer or receiving hemopoietic stem cell transplant (HSCT) at G.Gaslini Children Hospital, Genoa, Italy. From 2006 to 2008, 463 children were treated for a malignancy or received a HSCT for a non-neoplastic disease. The Table reports on the 30 cases of pneumonia observed. In the 3 (10%) cases due to M. pneumoniae, two in patients with non-Hodgkin lymphoma and one with acute myelogenous leukemia, the pathogen was detected by PCR on throat swabs. In all cases the onset of the disease was represented by febrile granulocytopenia with symptoms of pneumonia (14% of pneumonias in granulocytopenic patients), with lung infiltrates (Fig. 1), in two cases bilateral (Fig. 1, panels B,C) at CT scan. All patients were receiving co-trimoxazole for P. jiroveci prophylaxis. Empirical antibacterial (piperacillin-tazobactam) and antifungal (liposomal amphotericin B) therapy were administered, associated with clarithromycin, according to our protocol for patients with severe pneumonia, until the availability of specific diagnosis [6]. In all cases galactomannan antigen resulted negative and antifungals were discontinued at documentation of M. pneumoniae infection. Antimycoplasma therapy was administered for 21 days and all patients survived without sequelae. In granulocytopenic cancer patients, fungal pneumonia represents a serious complication because of its high mortality. CT imaging may be not specific, since a halo sign is present very early and for a short period of time and in the subsequent days radiological features of IFD may be aspecific [7]. According to the most recent classification [8], a possible pulmonary IFD is defined by the presence of appropriate host factors in a patient with fever, respiratory symptoms, chest CT scan with dense, well-circumscribed lesions with or without a halo sign, in absence of mycological support. Even if the definitions of IFD should be used for research purposes only [8], actually they are adopted in the everyday clinical practice. The three cases we described would have fulfilled the criteria for a possible IFD, if we had not checked for M. pneumoniae infection, and it must be stressed that they represented 1/3 of cases of pneumonias that we could have classified as possible IFD. In these cases also the possibility of a polymicrobial infection, including Aspergillus, should be considered [9,10]. Our report indicates that M. pneumoniae should be included in the differential diagnosis of pneumonia in granulocytopenic children since it requires specific therapy.

Development and validation of a multiplex quantitative polymerase chain reaction assay for the detection of Mollicutes impurities in human cells, cultured under good manufacturing practice conditions, and following European Pharmacopoeia requirements and the International Conference on Harmonization guidelines

BACKGROUND AIMS The clinical applications of in vitro manipulated cultured cells and their precursors are often made use of in therapeutic trials. However, tissue cultures can be easily contaminated by the ubiquitous Mollicutes micro-organisms, which can cause various and severe alterations in cellular function. Thus methods able to detect and trace Mollicutes impurities contaminating cell cultures are required before starting any attempt to grow cells under good manufacturing practice (GMP) conditions. METHODS We developed a multiplex quantitative polymerase chain reaction (qPCR) assay specific for the 16S-23S rRNA intergenic spacer regions, for the Tuf and P1 cytoadhesin genes, able to detect contaminant Mollicutes species in a single tube reaction. The system was validated by analyzing different cell lines and the positive samples were confirmed by 16S and P1 cytoadhesin gene dideoxy sequencing. RESULTS Our multiplex qPCR detection system was able to reach a sensitivity, specificity and robustness comparable with the culture and the indicator cell culture method, as required by the European Pharmacopoeia guidelines. CONCLUSIONS We have developed a multiplex qPCR method, validated following International Conference on Harmonization (ICH) guidelines, as a qualitative limit test for impurities, assessing the validation characteristics of limit of detection and specificity. It also follows the European Pharmacopoeia guidelines and Food and Drug Administration (FDA) requirements.