Francesca Lippi

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.

Influence of plant genotype on the selection of nodulating Sinorhizobium meliloti strains by Medicago sativa

We analysed the genetic diversity of 270 Sinorhizobium meliloti strains isolated from nodules of three different Medicago sativa varieties, planted in three different Italian soils, combining the Analysis of Molecular Variance (AMOVA) with the Random Amplified Polymorphic DNA (RAPD) technique to estimate variance among RAPD patterns with the aim to draw an objective description of the population genetic structure. Results indicated that a general intraspecific genetic diversity was globally distributed among all the population, however a very high level of diversity was found among strains nodulating different Medicago sativa varieties. Moreover the distribution of the RAPD haplotypes among the plant varieties also showed to be non-random. The overall data indicated that the plant genotype is a major factor in shaping the genetic structure of this natural Rhizobium population.

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.

Distribution of invasive meningococcal B disease in Italian pediatric population: Implications for vaccination timing

Neisseria meningitidis group B (MenB) is a leading cause of meningitis and sepsis. A new vaccine has been recently licensed. The aim of the present study was to evaluate the epidemiology of MenB disease in pediatric age and define the optimal age for vaccination. All patients aged 0-18 years admitted with a diagnosis of meningitis or sepsis to the 83 participating Italian pediatric hospitals were included in the study. Blood and/or cerebrospinal fluid (CSF) samples were tested by Realtime-PCR and/or culture. One hundred and thirty-six cases (mean age 5.0 years, median 2.7) of MenB disease were found. Among these, 96/136 (70.6%) were between 0 and 5 years, 61/136 (44.9%) were between 0 and 2 years. Among the latter, 39/61 (63.9%) occurred during the first year of life with highest incidence between 4 and 8 months. A case-fatality rate of 13.2% was found, with 27.8% cases below 12 months. Sepsis lethality was 24.4%. RT-PCR was significantly more sensitive than culture: 82 patients were tested at the same time by both methods, either in blood or in CSF; MenB was found by RT-PCR in blood or CSF in 81/82 cases (98.8%), culture identified 27/82 (32.9%) infections (Cohens Kappa 0.3; McNemars: p<10⁻⁵). The study shows that the highest incidence of disease occurs in the first year of age, with a peak between 4 and 8 months of life; 30% of deaths occur before 12 months. The results suggest that the greatest prevention could be obtained starting MenB vaccination in the first months of life; a catch-up strategy up to the fifth year of life could be considered. Our results also confirm that Realtime PCR is significantly more sensitive than culture. In those countries where only isolate positive infections are counted as cases, the incidence of MenB infection results highly underestimated.

The inclusion of ADA-SCID in expanded newborn screening by tandem mass spectrometry.

Severe combined immunodeficiency due to adenosine-deaminase defect (ADA-SCID) is usually deadly in childhood because of severe recurrent infections. When clinical diagnosis is done, permanent damages due to infections or metabolite accumulation are often present. Gene therapy, bone marrow transplantation or enzyme replacement therapy may be effective if started early. The aim of this study was to set-up a robust method suitable for screening with a minimized preparation process and with inexpensive running costs, for diagnosing ADA-SCID by tandem mass spectrometry. ADA-SCID satisfies all the criteria for inclusion in a newborn screening program. We describe a protocol revised to incorporate adenosine and 2-deoxyadenosine testing into an expanded newborn screening program. We assessed the effectiveness of this approach testing dried blood spots from 4 genetically confirmed early-onset and 5 delayed-onset ADA-SCID patients. Reference values were established on 50,000 healthy newborns (deoxyadenosine <0.09μmol/L, adenosine <1.61μmol/L). We also developed a second tier test to distinguish true positives from false positives and improve the positive predictive value of an initial abnormal result. In the first 18 months, the pilot project has identified a newborn with a genetically confirmed defect in adenosine deaminase (ADA) gene. The results show that the method having great simplicity, low cost and low process preparations can be fully applicable to a mass screening program.

Evaluation of bivalent human papillomavirus (HPV) vaccine safety and tolerability in a sample of 25 year old Tuscan women

The aim of this study was to gather data on the safety of the HPV-16/18 AS04-adjuvated vaccine among women aged 25, evaluating the frequency and severity of adverse events reported after vaccination and to compare the results obtained with previously published data regarding a sample of Italian preadolescents. Every woman residing in the province of Florence and in the age group targeted by the cervical cancer screening was invited to participate. Participants registered daily, for 14 d post-vaccination, solicited local and systemic reactions, as well as unsolicited adverse events in a developed ad hoc safety diary card. Data were collected in a database in Access and analyzed using STATA 11 SE statistical software. A total of 271 participants were recruited in the study group. All three diary cards were completed and delivered by 186 subjects (85.7% of participants). In all, a total of 616 diary cards were collected: 216 after the 1st dose, 209 after the 2nd dose and 191 after the 3rd dose. No severe symptoms were registered. The most frequently reported adverse reaction proved to be pain at the site of injection (83.4% of doses), followed by local swelling (20.8%) and pyrexia (14.6%). The safety and tolerability of the HPV-16/18 AS04-adjuvated vaccine in this sample of adult women aged 25 did not differ much from that previously observed in a sample of preadolescents Italian girls. Fever and local pain were however more frequently registered in our sample of adult women.

Evans syndrome and antibody deficiency: an atypical presentation of chromosome 22q11.2 deletion syndrome

We report a case of an 8-year-old male patient with Evans syndrome and severe hypogammaglobulinemia, subsequently in whom the 22q11.2 deletion syndrome (22q11.2 DS) was diagnosed. No other clinical sign of 22q11.2 DS was present with the exception of slight facial dysmorphism. The case is of particular interest because it suggests the need to research chromosome 22q11.2 deletion in patients who present with autoimmune cytopenia and peculiar facial abnormalities, which could be an atypical presentation of an incomplete form of 22q11.2 DS.

Newborn screening for PIDs using both TREC and KREC identifies late occurrence of B cells

Tuscany is the first region in Italy to have implemented a neonatal screening for congenital immunodeficiencies using both tandem mass spectrometry for early and late-onset adenosine deaminase and purine-nucleoside phosphorylase deficiency (1) and multiplex Real-Time PCR for TREC and KREC quantification on Dried Blood Spots (DBS) (2). The screening program with TREC and KREC started on December 2013 and, basing on the last data update of March 2017, it has screened a total of 18981 newborns in these first 3 years. We have had no cases of low or diminished TRECs but 5 cases of low/absent KRECs. This article is protected by copyright. All rights reserved.

Cutaneous PAN and Common Variable Immunodeficiency: what causes what?

Background Cutaneous polyarteritis nodosa (c-PAN) is a necrotizing vasculitis of small and medium-sized vessels limited to the skin, characterized by the presence of subcutaneous nodular, painful, non purpuric lesions with or without livedo reticularis occurring predominantly in the lower extremities, with no systemic involvement. The cause of c-PAN is unknown: infectious agents in childhood and immunodeficiency in adults have been associated with the disease. Aim To our knowledge the association of c-PAN and Common Variable Immunodeficiency (CVID) has not been reported so far. Case report A 2 year-5 months old girl was referred for intermittent fever and cutaneous erythematous, painful nodular lesions on feet, ankles and pretibial regions. A skin biopsy showed a necrotizing non-granulomatous vasculitis. Diagnosis of cutaneous polyarteritis nodosa was made and treatment with methotrexate (MTX), prednisone and ibuprofen was started. One year later, due to insufficient response, MTX was switched to azathioprine. We obtained a good clinical control in 4 months, thus NSAIDs and steroids were progressively stopped. At an 8 months follow-up, laboratory test were all in the normal range, except for progressive hypogammaglobulinemia (IgA and IgM, 29.4 mg/dl; and 28 mg/dl; IgG= 571 mg/dl). Immunological tests showed: IgG2 deficiency (42 mg/dl) total white count at lower limits, with lymphopenia (21.6%); lymphocyte subpopulations showed deficiency of CD19+ B cells (3%, normal values: 6-25%) and a poor response to protein vaccines. Due to the 1-year persistent remission on therapy, AZA was tapered and stopped. After several months, hypogammaglobulinemia persisted, thus excluding a potential side effect related to AZA treatment. Six months later, due to a disease flare, AZA was restarted in addition to prednisone (1 mg/kg/die) and IVIG every 4 weeks (2 gr/ kg). IVIG treatment was added in order to combine their anti-inflammatory effect with the CVID replacement therapy. At last follow-up her c-PAN is in clinical remission on AZA only. IVIG treatment is still needed for replacement (400 mg/kg monthly), due to her CVID. Of note, during her long term (6 years) follow-up in our Unit, she never developed any major infection. Conclusions c-PAN and CVID are both due to immune system dysregulation, with not well known mechanisms. In our patient, immunosuppressive therapy could have induced a B-immunodeficiency, or could have uncovered a preexisting immunodeficient status. However, a casual association or the possibility of c-PAN as onset of CVID cannot be excluded.