VALUTAZIONE DELL'IMPIEGO DI TAMPONI SALIVARI SECCHI (DSS, DRIED SALIVA SWAB) PER LA DIAGNOSI E LO SCREENING NEONATALI DELL'INFEZIONE CONGENITA DA CYTOMEGALOVIRUS.

Background 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.