Francesca Bonvicini

Recent developments in rapid multiplexed bioanalytical methods for foodborne pathogenic bacteria detection

AbstractFoodborne illnesses caused by pathogenic bacteria represent a widespread and growing problem to public health, and there is an obvious need for rapid detection of food pathogens. Traditional culture-based techniques require tedious sample workup and are time-consuming. It is expected that new and more rapid methods can replace current techniques. To enable large scale screening procedures, new multiplex analytical formats are being developed, and these allow the detection and/or identification of more than one pathogen in a single analytical run, thus cutting assay times and costs. We review here recent advancements in the field of rapid multiplex analytical methods for foodborne pathogenic bacteria. A variety of strategies, such as multiplex polymerase chain reaction assays, microarray- or multichannel-based immunoassays, biosensors, and fingerprint-based approaches (such as mass spectrometry, electronic nose, or vibrational spectroscopic analysis of whole bacterial cells), have been explored. In addition, various technological solutions have been adopted to improve detectability and to eliminate interferences, although in most cases a brief pre-enrichment step is still required. This review also covers the progress, limitations and future challenges of these approaches and emphasizes the advantages of new separative techniques to selectively fractionate bacteria, thus increasing multiplexing capabilities and simplifying sample preparation procedures. FigureNew analytical formats are under development to allow multiplexed detection of foodborne pathogens, thus cutting assay times and costs and enabling large scale screening procedures. A variety of analytical strategies are being explored to reach this goal. This review covers the recent progresses, limitations and future challenges of these approaches

Portable device based on chemiluminescence lensless imaging for personalized diagnostics through multiplex bioanalysis.

A simple and versatile analytical device designed to perform, even simultaneously, different types of bioassays has been developed and optimized. A transparent microfluidics-based reaction chip, where analytes were quantitatively detected by means of biospecific reactions and chemiluminescence detection, was placed in contact with a thermoelectrically cooled CCD sensor through a fiber optic taper. Such a lensless contact imaging configuration combined adequate spatial resolution and high light collection efficiency within a small size portable device. The miniaturization of the reaction chamber ensured short analysis times (in the minutes range), while the use of chemiluminescence detection provided wide signal dynamic range and high detectability, down to attomole levels of protein and femtomole levels of nucleic acid analytes. A model hybrid panel test was realized by combining an enzyme assay for alkaline phosphatase activity, a nucleic acid hybridization assay for Parvovirus B19 DNA, and an immunoassay for horseradish peroxidase as a model antigen. The successful simultaneous quantification of the three targets demonstrated that a range of analytes, from enzymes to antigens, antibodies, and nucleic acids, can be measured in a single run, thus enabling the realization of a complete, personalized diagnostic panel test for early diagnosis of a given disease and patient follow-up.

Human papillomavirus DNA testing by PCR-ELISA and hybrid capture II from a single cytological specimen: concordance and correlation with cytological results.

BACKGROUND AND OBJECTIVES A persistent infection by high-risk HPV is now considered as the major cause of cervical carcinoma. The use of a single cytological specimen for HPV DNA testing by two different molecular methods was analyzed and validated. STUDY DESIGN HPV DNA testing by PCR-ELISA and hybrid capture II HPV test (HC-II), was investigated on 317 cytological samples obtained from Italian women. Two hundred twenty-seven women were referred to virological lab for HPV DNA testing during cytological routine screening and 90 during a cytological and virological follow-up after a conization or hysterectomy. RESULTS Overall, the concordance between the two assays was high (K=0.87). Compared with PCR-ELISA, the HC-II showed a sensitivity of 91.7% and a specificity of 95.4%. Although the analytical sensitivity of the PCR-ELISA was higher, the performance of the two tests did not differ in recognizing HPV DNA positive patients with either low or high-grade squamous intraepithelial lesions (LSIL or HSIL). HPV DNA positivity was directly correlated with the severity of cytological diagnosis (P<0.005). CONCLUSIONS In view of the comparable results obtained with the two assays and of the ease of use, and higher throughput of HC-II, it seems advisable, with a single cytological specimen, to employ the HC-II test as a first-line assay, either for screening or diagnosis, and to perform reflex PCR on positive samples, if typing of prevalent high risk HPVs is needed.

Parvovirus B19 DNA Is Commonly Harboured in Human Skin

Background: Parvovirus B19 is the aetiological agent of erythema infectiosum. The presence of B19 DNA in lesional skin of other cutaneous manifestations has frequently been reported although there is disagreement on the role of the B19 virus in tissues. Objectives: To investigate the presence of B19 DNA (1) in skin lesions of patients with a described B19-related disease, (2) in skin lesions of B19-unrelated diseases and (3) in healthy skin. Methods: A total of 121 skin samples were examined for the presence of B19 DNA by PCR assays and peptide-nucleic-acid-based in situ hybridisation techniques. Results: B19 DNA was detected in 11/38 (28.9%) pityriasis lichenoides, 8/30 (26.7%) melanocytic naevi, 5/29 (17.2%) primary melanomas and 6/24 (25.0%) healthy skin biopsies. A difference in B19 DNA prevalence was observed in specimens grouped according to age, irrespective of pathologies. Conclusions: B19 DNA can be found in skin tissues of patients with pityriasis lichenoides as well as in lesions not related to B19 infection and in healthy controls. B19 DNA can be detected in skin of young subjects in a significantly high rate compared to adults, suggesting that viral persistence may be the usual outcome after primary infection.

Placental endothelial cells can be productively infected by Parvovirus B19

BACKGROUND Parvovirus B19 vertical transmission occurs in about 30% of cases of maternal infection and may result in foetal hydrops or intrauterine foetal death. Details on the mechanism of transplacental transmission of B19 virus and subsequent foetal infection have not been elucidated. OBJECTIVE To investigate the extent and distribution of B19 virus infection in placental tissues. STUDY DESIGN Virological, histological, electron microscopy, immunohistochemical and immunofluorescence analysis of placental tissues obtained from a case of intrauterine foetal death caused by B19 virus. RESULTS Real-time PCR analysis showed B19 virus DNA in placental samples. Histology, immunohistochemistry and electron microscopy demonstrated the concomitant infection of both foetal erythroid precursors and placental endothelial cells. In situ hybridisation for B19 virus nucleic acids, immunohistochemistry for B19 virus proteins and double labelling immunofluorescence confirmed that endothelial cells were productively infected by B19 virus. CONCLUSION Foetal capillary endothelium in placental villi can be an additional target of productive B19 virus infection. Infection of placental endothelial cells may lead to a structural and functional damage critical both for altering maternal-foetal blood exchanges and for spreading the infection to the foetus, possibly concurring to the development of foetal hydrops and intrauterine foetal death.

Diagnosis of fetal parvovirus B19 infection : value of virological assays in fetal specimens

Objective  The purpose of our work was to examine the most reliable laboratory diagnosis of fetal parvovirus B19 infection in hydropic fetuses by evaluating the most appropriate clinical sample and laboratory test.

Diagnostic procedures in B19 infection

In immunologic normal hosts, both children and adults, B19 can cause acute, generally self-limiting diseases. The infection leads to a viremia that can be present, at high titre, for about one week, then the onset of a specific immune response controls the infection. B19 infection in pregnancy can be associated with non-immunologic foetal hydrops or foetal death. In immunocompromised hosts, B19 can persist over several months and sometimes years. Persistent or recurrent B19 infections can be associated with chronic clinical manifestations or with transient clinical syndromes, generally related to the recrudescence of viral replication. Since the infection has been associated with a wide variety of clinical manifestations and some clinical features of B19 infection, such as anemia, artropathy and rash, can be common to other pathogens, a laboratory diagnosis of B19 infection is required. A diagnostic protocol must consider both the type of pathology and the type of patient. In immunocompetent individuals serological and virological testing is complementary, while in immunocompromised patients viral detection is the diagnosis of choice. Viral detection methods are generally based, nowadays, on the direct detection of B19 genome in clinical specimens. B19 DNA is mainly detected by hybridizations assays and by the most sensitive PCR assays. Serological diagnosis of B19 infection is generally achieved by detection of IgM and IgG antibodies to the B19 structural proteins VP1 and VP2. IgM detection is most often performed by capture assays, both in EIA and RIA formats, IgG are mainly detected by indirect EIA and immunofluorescence tests.

Parvovirus B19 in pregnancy: possible consequences of vertical transmission

The aim was to determine the outcome of pregnancies complicated by maternal Parvovirus B19 (B19) infection.

Cationic-anionic polyelectrolyte interaction as a tool to graft silver nanoparticles on hydroxyapatite crystals and prevent cytotoxicity

This paper reports an easy, green and low cost method to support silver nanoparticles (AgNPs) onto functionalized hydroxyapatite (HA) crystals. The process involves low molecular weight poly(ethylenimine) (PEI) as a reducing and stabilizing agent of AgNPs and polyacrylic acid (PAA) as a functionalizing agent of hydroxyapatite (HAPAA). With respect to HA, the negatively charged surface of HAPAA significantly improves the interaction with the positively charged surface of PEI-stabilized AgNPs. AgNPs content up to about 6.5 wt% in the composite HAPAA–AgNPs crystals has been obtained by varying the volume of the AgNPs suspension submitted to interaction with HAPAA. The cytotoxicity of the crystals, evaluated through in vitro tests on MG63 osteoblast-like human cells, increases on growing Ag content, but it is reduced by the presence of PAA. The HAPAA–AgNPs crystals display similar dose-dependent antibacterial activity towards gram-positive and gram-negative bacteria. The comparison of data indicates that an AgNPs content of about 2 wt% allows for combining absence of cytotoxicity with a significant long-standing antibacterial activity towards both Staphylococcus aureus and Escherichia coli.

Keeping Pace with Parvovirus B19 Genetic Variability: a Multiplex Genotype-Specific Quantitative PCR Assay

ABSTRACT Three genotypes have been identified within the parvovirus B19 species (B19V), and such genetic diversity may have significant implications for the development of molecular detection assays. In the present study, B19V genetic variability has been examined on a subset of genomic sequences available in the NCBI nucleotide database, and a quantitative PCR (qPCR) assay able to detect, differentiate, and quantify all viral variants has been established. The designed primers and probes have been used for the development of alternative detection formats, based on a combined use of intercalating dye and genotype-specific hydrolysis probes. The qPCR assay analytical performances have been determined on the 1st WHO International Reference Panel for Parvovirus B19 Genotypes. The developed qPCR protocols allow for the detection of genotypes 1 to 3 with equal accuracy, and with a limit of detection (LOD) of 200 IU/ml. A comparison of routine performance was carried out with respect to a previously established assay specifically validated on B19V genotype 1. For 130 clinical samples analyzed, 126 showed concordant results (31 positive and 97 negative), while 4 showed discordant results. Overall, the genotype-specific qPCR assay showed a sensitivity of 93.94% and a specificity of 97.94%, with an agreement rate of 96.92%. The proposed qPCR assay and the alternative protocols developed, each with robust performance, may allow choice with respect to operational systems and diagnostic requirements and might contribute to provide a more reliable diagnostic service and epidemiological surveillance of B19 virus.