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VALUTAZIONE DELL'IMPIEGO DI TAMPONI SALIVARI SECCHI (DSS, DRIED SALIVA SWAB) PER LA DIAGNOSI E LO SCREENING NEONATALI DELL'INFEZIONE CONGENITA DA CYTOMEGALOVIRUS.
Get PDFBackground
The identification of infected newborns at birth is necessary to prevent, or at least reduce, possible serious damages due to congenital Cytomegalovirus infection (cCMV). Viral isolation assay on saliva and urine specimens collected until the 14th day of birth is considered the gold standard method for cCMV’s diagnosis, but it is a slow method and it needs specialized laboratories. Easy and inexpensive collection, handling and processing of samples are important for implementation of neonatal screening. The aim of this PhD project is to develop a method which could meet the requirements of a screening test: low cost (sampling, reagents and workload) with high sensibility and specificity; the second purpose is to identify a possible factor of poor prognosis.
Material and Methods
In this study dried saliva swabs or DSS, a nylon-flocked saliva swab (Copan) without any medium, were used to collect clinical specimens. The study was divided in four phases, each one divided in two steps: validation and clinical tests. In the 1st phase 410 DSS were collected from 21 babies with cCMV (follow-up group) and 365 from newborns or children who were in Mangiagalli’s Hospital on July 2008 (random group). All DSS were extracted by commercial kit and the results were compared to classical saliva swabs or CSS collected and stored in Viral transport medium (VTM) at 4°C. CSS were tested by rapid viral isolation (IR-p72) and nested-PCR in house (n-PCR). In the 2nd phase an extraction in-house was performed on DSS, and dried swabs were re-hydrated with E-MEM (cell’s growth medium). 192 DSS were collected: 34 from a follow up group, 141 from children who attending preschool and 17 from babies with suspect of infection. All results were compared between DSS just agitated by vortex or extracted by thermal shock (ts) and CSS tested by nested-PCR. In the 3rd phase a commercial Real Time-PCR (RT-PCR) was performed for DSS vortexed or extracted by ts. Previously poor results were obtained with samples in E-MEM, therefore molecular grade water was preferred to re-hydrating DSS. 64 dried saliva swabs were collected from 45 follow-up children and 14 babies with suspect of infection. Results were compared between DSS tested in RT-PCR or n-PCR and CSS in n-PCR. In the 4th phase a genotyping methods were performed on DSS: RFLP (Restriction Fragment Length Polymorphism) for gB gene and, by collaboration with a Dutch group from LUMC (Leids Universitair Medisch Centrum), a Real Time-PCR in–house for genes gB and gH. So 101 DSS were collected and tested from follow up group’s children and suspected of infection.
Results
In every phase the sensibility between DSS and CSS was 100% regardless of treatment and studied groups. The specificity was 93% between DSS and CSS tested by IR-p72; it was higher if compered to CSS in n-PCR. In follow-up group the specificity was 58 and 70% between DSS and CSS in n-PCR. The most frequent genotype was gB1 both in RFLP (38%) and RT-PCR (34%). The most frequent gH strain was gH1 (48%). 19% of samples had mixed genotype of gB or gH or both; 3 patient (3%) had three gB strain in the same time.
Conclusion
Dried saliva swab is a good tool to detect CMV infection. Despite the treatments Low specificity in follow-up group is a consequence of high sensibility of DSS-test. In fact the babies of the follow up group had CMV infection, therefore the positive results might have been no false. Real Time-PCR (Argene) on DSS treated in molecular biology grade water gave optimal results. Pre-PCR treatments (vortexing or vortexing plus thermal shock), seem to have no influence. Genotyping from DSS by Real time-PCR could be a good alternative to genotyping from DBS (dried blood spots), because saliva has higher viral load. Confirmation of these data in a larger study will indicate that Real Time-PCR DSS testing (treat adding grade water and just vortexing), being simple and cheap , could be a suitable method for a neonatal cCMV infection screening
Valutazione della protezione immunitaria verso poliovirus in soggetti in regime di adozione internazionale
No full textIntroduzione. Il processo di eradicazione della polio non ha ancora raggiunto l’obbiettivo a più di 25 anni dal suo lancio. Nonostante i notevoli risultati, persistono Paesi endemici verso Poliovirus (PV) ed altri in cui si verificano reintroduzioni risultanti in epidemie, laddove il livello di protezione immunitaria della popolazione non è adeguato. L’Italia accoglie ogni anno circa 4,000 bambini in regime di adozione internazionale provenienti anche da Paesi extracomunitari; il loro stato immunitario nei confronti del PV è a volte sconosciuto o dubbio. Questo studio preliminare ha avuto lo scopo di verificare i livelli di immunizzazione antipoliomelitica di soggetti in regime di adozione internazionale in Nord-Italia.
Metodi. La determinazione del titolo anticorpale è stata effettuata con il test di microneutralizzazione in accordo con OMS (WHO/EPI/GEN/93.9). I sieri diluiti da 1:4 a 1:4096 dei soggetti erano saggiati contro 100DC50 dei tre sierotipi di PV in cellule Hep2-C. La più alta diluizione di siero in grado di neutralizzare l’effetto citopatico in coltura costituiva il titolo anticorpale. Secondo le indicazioni OMS è ritenuto protettivo un titolo ≥ 1:8.
Risultati. Sono stati arruolati 59 soggetti (6m-17a) provenienti da diversi Paesi (Asia, Africa, Est-Europa). Considerando i singoli sierotipi, circa il 19%, il 7% ed il 20% degli esaminati mostravano livelli immunitari non-protettivi rispettivamente verso PV1, PV2 e PV3.
Conclusioni. Per garantire l’eradicazione della polio è fondamentale monitorare lo stato vaccinale della popolazione, sia autoctona che di recente introduzione. In questo studio si sono evidenziati bambini nei quali i livelli immunitari non erano sufficienti. Questo richiama l’attenzione sia sul grado di protezione immunitaria, sia sull’attendibilità della documentazione vaccinale che li accompagnava e quindi sulla necessità di mettere in atto, al loro ingresso in Italia, le misure più efficaci di adeguamento immunitario
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Comparison of three real-time pcrs for the quantification of CMV-DNA on dbs
No full textBRIEF INTRODUCTION
The Dried Blood Spot (DBS) is a useful sample for diagnosing congenital CMV infection (cCMV). Some
authors suggest that a high viral load in blood samples could have an unfavourable prognostic value.
In this study we compared the performances of three methods in measuring CMV viral load in DBS.
MATERIALS AND METHODS
We tested 36 DBS samples from QCMD panels (2006-2009). Purification of DNA was performed with a
commercial kit (QIAGEN), following the DBS protocol. Viral DNA was quantified by means of an
in-house method (gB gene) and two commercial kits “CMV, HHV6, 7, 8 R-gene”, Argene (for CMV:
ppUL83) and “Q-CMV Real-time Complete kit”, Nanogen (MIEA).
CLINICAL CASES OR SUMMARY RESULTS
Intra- and inter- assay viral load values were within ±0.5log10 unit. All DBS prepared with whole
blood spiked with 1E+02 copies/ml of CMV-DNA tested negative, while those with viral loads of
1E+03 copies/ml gave positive results in about half of samples.
CONCLUSIONS
The results obtained in this study are similar to datasets submitted by QCMD participants. All three
methods detect equally high viral loads in DBS but they should be improved to identify also lower
ones. The “CMV, HHV6, 7, 8 R-gene” kit seems to be particularly interesting, as it provides a readyto-
use and cheap mix which permits also the simultaneous detection of different herpesviruses
Two case of Human Parechovirus 3 (hPEV3) infection in neonatal encephalitis
No full textTwo case of Human Parechovirus 3 (hPEV3) infection in neonatal encephaliti
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