We describe a simple, sensitive, and reproducible method for using whole blood collected onto filter paper (dried blood spots) for detection and genotyping of hepatitis C virus RNA that can be useful in large field studies, particularly in settings where collection, preparation, storage, and shipment of samples at controlled temperature can be difficult.Dried blood spots (DBS) have been used worldwide for the neonatal screening of congenital disorders (2, 8). Seroepidemiological studies have been conducted on DBS residual to neonatal screening to assess human immunodeficiency virus (HIV) prevalence among childbearing women (9, 10). Recently, several studies were focused on DBS for detecting drug resistance mutations (6) and for tracking global spreading of HIV type 1 subtypes (4) in proviral HIV DNA. However, RNA is notoriously less stable, and standardization of DBS for viral RNA detection would be of great benefit for application to large field studies, since DBS collection is easy, does not require skilled phlebotomists and expert technicians, and is suitable for storage and shipment to laboratory in settings where these issues are problematic. Recently, dried plasma spots and DBS have been used for HIV RNA detection and quantification, showing good correlation with titers obtained with conventional plasma samples (5, 11). However, these observations were limited to short storage at room temperature (11) or at 37°C (5), and a loss of viral titers occurred during storage.For hepatitis C virus (HCV) RNA detection on dried spot samples, the available data are much less exhaustive. A complete match between frozen serum and dried plasma spots, though with a loss of titers after room temperature storage, has been observed (1).This study was aimed at developing a simple, sensitive, and reproducible method for using DBS in HCV RNA detection and genotyping.The study complied with all relevant national guidelines and institutional policies. Residual laboratory samples of EDTAwhole blood of 39 HCV antibody (Ab)-positive and 16 HCV Ab-negative patients, undergoing routine hematological controls, were used. HCV Abs were determined by third-generation assay (Abbott Diagnostics). Among the Ab-positive patients, 34 had HCV RNA levels ranging between 9,640 and 5,100,000 IU/ml (Amplicor HCV Monitor; Roche Molecular Systems Inc.), and 5 were HCV RNA negative (Versant HCV TMA; Bayer Diagnostic Inc.). The HCV genotype was known for eight patients (four had 1b; two had 2a/2c; one had 3a; and one had 4c/4d).DBS were realized within 5 h from venipuncture by carefully spotting, in multiple replicates for each patient, 50 l of EDTA-whole blood on SS grade 903 filter paper (Schleicher & Schuell Inc.). Two DBS from each patient were pooled and processed for each assay.HCV RNA detection was performed with both in-house reverse transcriptase PCR (RT-PCR) and transcription-mediated amplification (TMA). For RT-PCR, RNA extraction was performed with Boom technology, utilizing silica-based RNA isolation (3), which was purchased from Organon ...

The number of children born to an HIV-positive mother calls for continued efforts to prevent vertical transmission of the infection.

No abstract

To describe the dynamic of HIV-1 prevalence in Italian childbearing women and to estimate the future incidence of pediatric AIDS due to vertical transmission, dried-blood specimens collected from a consecutive sample of newborns in all Italian regions were examined for the presence of anti-HIV-1 antibodies (HIV-Ab) after the routine neonatal screening program was completed. Of 555,722 blood samples collected and examined for HIV-Ab between 1990 and 1993, 550 (0.099%) were positive. Nationwide, the HIV seroprevalence decreased between 1990 and 1992 (0.124% in 1990, 0.100% in 1991, 0.085% in 1992), and increased, as compared with that in the previous year, in 1993 (0.096%). In an univariate analysis, HIV seroprevalence was positively was positively associated with being born in regions having higher AIDS cumulative incidence and in metropolitan areas, but negatively associated with year of delivery. In a multiple logistic regression analysis, only the AIDS cumulative incidence level of the delivery area and being born in a metropolitan area remained independently associated with HIV seroprevalence. Our results show significant geographical variation in the spread of HIV infection among childbearing women in Italy and provide useful indications to target prevention and care strategies for HIV-infected women and their children and to estimate the potential impact of implementing programs aimed at preventing vertical transmission of HIV infection.

A prevalence assessment of HIV infection among parturients was performed in order to estimate the future incidence of pediatric AIDS cases and obtain data on the trend of the infection in Italy. Consecutive whole blood samples from newborns collected on filter paper from all regions of Italy (October-December 1990) for routine metabolic screenings, were anonymously examined for HIV-1 antibodies by an ELISA technique. Positive results were confirmed by Western blot. Among 97,658 blood samples tested, 121 (0.124%, 95% confidence interval Poisson distribution 0.103-0.148) were positive. A high prevalence of HIV infection (> 0.2%) was observed in four regions from North and Central Italy while, in some regions in the South, the seroprevalence was ten times lower. In the Lazio Region, namely in Rome, 5 inner-city hospitals with a particularly high prevalence of HIV infection were identified. Assuming that the prevalence of HIV infection observed in the population in this study may reflect the infection rate in the total population of childbearing women, we estimate that approximately 700 newborn babies were delivered from HIV-positive mothers in Italy during 1990. The identification of high prevalence areas could allow for the development of "targeted" testing programs to plan and provide adequate counselling and care of HIV-infected women and their infants.