Monica Musiani - Academia.edu (original) (raw)

Papers by Monica Musiani

Journal of Clinical Pathology, Oct 1, 2007

Journal of Medical Virology, Aug 1, 2001

Adeno-associated viruses (AAV) are human parvoviruses that require helper function for their repl... more Adeno-associated viruses (AAV) are human parvoviruses that require helper function for their replication. Several studies have demonstrated that AAV DNA sequences can be found in the female genital tract but the incidence of infection seems very variable. A PCR-ELISA method detecting AAV DNA was developed for combining the speci®city and the sensitivity of conventional PCR with an objective interpretation of the results. In the PCR-ELISA, a de®ned number of cells from cervical specimens were digested and am-pli®ed with concomitant digoxigenin labeling. Digoxigenin-labeled ampli®ed products hybridized to a speci®c biotinylated probe were captured in streptavidin-coated microtiter wells by a biotin-streptavidin binding and were visualized by colorimetric immunoenzymatic reaction. PCR-ELISA was carried out in 110 cervical cytological specimens of women with or without the concomitant detectable presence of papillomavirus (HPV) DNA and the results were compared with those obtained by conventional PCR followed by dot blot hybridization. When compared to conventional PCR considered as reference standard, PCR-ELISA was found to be 98% sensitive and 96% speci®c. Out of the total 110 samples examined, 52.7% were positive for AAV DNA by both techniques, demonstrating a high prevalence of AAV infection in the uterine cervix. When analyzing samples with or without the presence of HPV DNA, 63.2 % of the samples were positive for HPV DNA and 41.5% of the samples were negative for HPV proved positive for AAV DNA by both PCR-ELISA and conventional PCR. Hence, PCR-ELISA , which can be completed in 1 day, proved to be a reliable method for an objective detection of AAV DNA in clinical samples. The present study showed a frequent infection of the cervical epithelium with AAV both in the presence and absence of HPV infection.

Luminescence, 2003

In this study we describe an efficient stable genetic transformation of the phytopathogenic bacte... more In this study we describe an efficient stable genetic transformation of the phytopathogenic bacterium Erwinia amylovora using a recombinant expression vector encoding the firefly luciferase gene of Photinus pyralis, which is further controlled by IPTG-inducible promoter. Stably transformed E. amylovora cells maintain the same infectivity as the wild-type strain and, after induction with IPTG, produce luciferase. Luminescence produced by the action of luciferase on an exogenous substrate was easily detectable by a simple and rapid bioluminescent assay (BL). The transformed E. amylovora strain maintains the same high emission level, even after passage in pears, until about 15 days post-infection. Our findings therefore show that the luciferase assay can be conveniently used to follow the bacterial movement in plant tissue and its dissemination in controlled environments.

Clinical Chemistry, Sep 1, 1999

Background: Quantitative PCR of viral nucleic acids can be useful clinically in diagnosis, risk a... more Background: Quantitative PCR of viral nucleic acids can be useful clinically in diagnosis, risk assessment, and monitoring of antiviral therapy. We wished to develop a chemiluminescence competitive PCR (cPCR) for parvovirus B19. Methods: Parvovirus DNA target sequences and competitor sequences were coamplified and directly labeled. Amplified products were then separately hybridized by specific biotin-labeled probes, captured onto streptavidin-coated ELISA microplates, and detected immunoenzymatically using chemiluminescent substrates of peroxidase. Chemiluminescent signals were quantitatively analyzed by a microplate luminometer and were correlated to the amounts of amplified products. Results: Luminol-based systems displayed constant emission but had a higher detection limit (100-1000 genome copies) than the acridan-based system (20 genome copies). The detection limit of chemiluminescent substrates was lower (20 genome copies) than colorimetric substrates (50 genome copies). In chemiluminescence cPCR, the titration curves showed linear correlation above 100 target genome copies. Chemiluminescence cPCR was positive in six serum samples from patients with parvovirus infections and negative in six control sera. Conclusions: The chemiluminescence cPCR appears to be a sensitive and specific method for the quantitative detection of viral DNAs.

Nat Protoc, 2007

PCR is an established technique providing rapid and highly productive amplification of specific D... more PCR is an established technique providing rapid and highly productive amplification of specific DNA sequences. The demand for equally rapid, sensitive and objective methods to achieve detection of PCR products has led to the coupling of PCR with ELISA. PCR-ELISA involves direct incorporation of labeled nucleotides in amplicons during PCR-amplification, their hybridization to specific probes and hybrid capture-immunoassay in microtiter wells. PCR-ELISA is performed in 1 d and is very flexible, with the ability to process simultaneously up to 96 or 384 samples. This technique is potentially automatable and does not require expensive equipment, and thus can be fundamental in laboratories without access to a real-time PCR thermocycler. PCR-ELISA has mainly been used to detect infectious agents, including viruses, bacteria, protozoa and fungi. A PCR-ELISA protocol for the qualitative detection of papillomavirus genomes and simultaneous typing of different genotypes are detailed here as an example of the technique.

Clinical Laboratory, Feb 1, 2002

1. Clin Lab. 2002;48(3-4):201-5. Humoral immune response to parvovirus B19 and serological diagno... more 1. Clin Lab. 2002;48(3-4):201-5. Humoral immune response to parvovirus B19 and serological diagnosis of B19 infection. Manaresi E, Gallinella G, Gentilomi G, Venturoli S, Zuffi E, Bonvicini F, Cricca M, Zerbini M, Musiani M. ...

Journal of Clinical Virology the Official Publication of the Pan American Society For Clinical Virology, Feb 1, 2004

Background and objectives: High-risk human papillomaviruses (HR-HPVs) are the primary cause of ce... more Background and objectives: High-risk human papillomaviruses (HR-HPVs) are the primary cause of cervical cancer. In order to meet with clinical requirements, a direct capture test with signal amplification (HC-II), able to detect the 13 prevalent HR-HPVs, has been developed and validated for cytological specimens. Study design: the use of HC-II assay with formaldehyde-fixed paraffin-embedded cervical biopsies, for retrospective studies or to support histological findings, was investigated by analysing three different sample treatments. The efficacy of this test was compared with a reference PCR-ELISA, using MY09/11 and GP5+/6+ consensus primers and the use of a single extraction method for both HC-II and PCR-ELISA assays was validated. Results: protease treatment of dewaxed biopsy sections allowed an optimal performance of HC-II and has also been validated for PCR-ELISA. Overall, on the analysis of 50 cervical samples HC-II and PCR-ELISA assays showed a high concordance (K = 0.80). Compared with PCR-ELISA, the HC-II had a sensitivity of 86.4% and a specificity of 92.9%. The results showed that short amplimers are necessary for a sensitive PCR-ELISA from formaldehyde-fixed paraffin-embedded biopsies, while HC-II showed a relatively low sensitivity for HPV18 within the HR probe pool. Conclusions: HC-II can be a valid tool for the diagnosis of HPV infection in biopsy material. The possibility to use the same specimen preparation material for both HC-II and PCR-ELISA allows HC-II positive specimens to be further processed by PCR-ELISA if specific genotyping is needed.

Journal of Virological Methods, Jun 30, 2009

The presence of HPV 16 E1 and E2 genes was detected in cervical cytological samples using polymer... more The presence of HPV 16 E1 and E2 genes was detected in cervical cytological samples using polymerase chain reaction assays. A total of 48 samples were analyzed from patients with HPV 16 infections associated with 13 low-grade cervical intraepithelial neoplasia and 35 high-grade cervical intraepithelial neoplasia. Disruption/deletion sites, within E1 and E2 genes, were detected using 6 primer pairs spanning the entire gene sequences. This technique is not able to recognize mixed DNA forms (integrated plus episomal DNA); therefore, it detects only the presence of pure integrated DNA. Both E1 and E2 genes were detected in 84.6% and in 62.9% of low and high-grade lesions, respectively. The rate of samples with disrupted/deleted genes was significantly higher in high-grade cervical intraepithelial neoplasia than in low-grade cervical intraepithelial neoplasia (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). In high-grade cervical intraepithelial neoplasia the disruption/deletion pattern involved both E1 and E2 genes and E2 gene was always involved, while in the low grade cervical intraepithelial neoplasia only E1 gene was involved. In conclusion, in high-grade cervical lesions E2 gene seems a suitable target to identify HPV 16 DNA integration into cellular genome.

Http Dx Doi Org 10 1081 Al 100002702, Feb 2, 2007

We used a luminescence-based biosensor to determine mercury in urine samples of subjects with or ... more We used a luminescence-based biosensor to determine mercury in urine samples of subjects with or without dental amalgam restorations. The system utilizes Escherichia coli as a host organism and firefly luciferase luc gene as a reporter under the control of the mercury-inducible promoter from the mer operon from transposon Tn21. The presence of mercury induces the specific gene sequence with consequent synthesis of the luciferase enzyme, and luciferin/luciferase-mediated light output occurs. The method showed a linear relationship between the light signal intensity and the Hg2+ concentration in the range of 1.67 × 10−13–1.67 × 10−7 M with a detection limit of 1.67 × 10−13 M, corresponding to 4.0 × 10−18 mol/tube. The system proved precise, accurate, and highly selective for mercury and methylmercury, with no light emission induced by other heavy metals; the matrix effect caused by urine components was minimal. The analysis of urine specimens showed that urinary mercury levels in subjects with amalgam fillings were slightly significantly higher than those in subjects without amalgam fillings (p < 0.1) and the statistically significant difference improved when creatinine-normalized values were considered (p < 0.05). In summary, from the analytical point of view this luminescent microbial biosensor represents an easy, rapid, and sensitive tool to analyse Hg2+ in biological and environmental samples. Along with the possibility of using 384-well microtitre plates requiring very low reagent volumes, these characterisitcs make the system suitable for the high-throughput screening of Hg2+ on large numbers of specimens. This method could help better clarify the relationship between dental amalgam and urinary mercury excretion by permitting large-scale analysis of many selected groups of subjects, with strict control of all the relevant parameters affecting Hg2+ concentration in urine, such as diet and environmental and occupational exposure.

Journal of Pharmaceutical and Biomedical Analysis, Dec 1, 1998

The development, analytical performance and applications of chemiluminescence imaging as a tool f... more The development, analytical performance and applications of chemiluminescence imaging as a tool for quantitative analyte localization in target biological specimens are described. The detection of acetylcholinesterase activity both in array format and on a target surface are described. A proposed application of the method is a 384 well microtiter format assay for high throughput screening of acetylcholinesterase inhibitors such as tacrine, a drug widely used in the treatment of Alzheimer's disease, and two recently developed analogues. The chemiluminescent system in conjunction with optical microscopy allowed localization of acetylcholinesterase in brain tissue sections. We also describe the chemiluminescent immunohistochemical localization of interleukin 8 in Helicobacter pylori infected gastric mucosa cryosections and an in situ hybridization assay for the detection of herpes simplex virus DNA in single cells.

Journal of Clinical Microbiology, Mar 1, 1996

Human parvovirus B19 infection in pregnancy represents a potential hazard to the fetus since feta... more Human parvovirus B19 infection in pregnancy represents a potential hazard to the fetus since fetal loss or fetal hydrops can occur. The risk of fetal loss due to transplacental B19 transmission has been evaluated in several studies using different diagnostic methods on maternal and fetal specimens. We analyzed the diagnostic value of virological and serological techniques on maternal serum, fetal cord blood, and amniotic fluid specimens obtained at the time of clinical diagnosis of fetal hydrops in 18 cases of B19 fetal hydrops. B19 DNA was detected by nested PCR, dot blot hybridization, and in situ hybridization assay. Anti-B19 immunoglobulin M and G antibodies were detected by immunoassays using recombinant B19 antigens. Our data suggest that for maternal sera, virological and serological methods have a complementary role in diagnosis, while for fetal specimens the in situ detection of B19 DNA in fetal cord blood is the most sensitive diagnostic system.

International Journal of Gynecological Pathology, 2008

A quantitative evaluation of p16 INK4A overexpression together with its topographical localizatio... more A quantitative evaluation of p16 INK4A overexpression together with its topographical localization in the epithelium of cervical biopsies from non-neoplastic lesions and cervical intraepithelial neoplasias (CIN 1, 2, and 3) was obtained by the development of an objective and sensitive immunohistochemical assay with chemiluminescent detection (CL IHC assay). The cervical biopsy samples were also checked for the presence of human papillomavirus nucleic acids. The quantitative evaluation of p16 INK4A expression was performed by combining 2 parameters: (1) intensity of the chemiluminescent-positive signal in the epithelium and (2) percentage of epithelium interested by the overexpression of p16 INK4A, to obtain a p16 INK4A expression score. A cut-off value was determined by using the receiver-operator characteristic analysis to distinguish between low-grade and high-grade CIN. Quantitative data showed that both p16 INK4A expression parameters increased with worsening grades of CIN and, when combined to obtain the p16 INK4A expression score, they showed a sharp discrimination among different lesions. The differences between the average p16 score of CIN1 versus CIN2 (0.57 versus 1.05), of CIN1 versus CIN3 (0.57 versus 1.31) and of CIN1 versus CIN2/3 (0.57 versus 1.20) were statistically significant. The quantitative evaluation of p16 INK4A expression by CL IHC assay could be therefore an interesting adjuvant method to distinguish different CIN grades and to predict the risk of progression of early CIN lesions.

Clinical Laboratory, Feb 1, 2006

Parvovirus B19 is a frequent contaminant of human blood and plasma derivatives and iatrogenic tra... more Parvovirus B19 is a frequent contaminant of human blood and plasma derivatives and iatrogenic transmission of B19 infection has been shown to occur through the administration of contaminated products. Manufacturing procedures, generally used for removal or inactivation of enveloped viruses (HIV, HCV and HBV) are not always effective in the elimination of B19 virus. A certain risk of contamination remains for some plasma derivatives due to the high-titer viral load in the starting blood donations and the extreme heat resistance and small size of the virus. This review provides an update on the different approaches currently available to detect, remove or inactivate B19 virus in order to enhance the safety margins of plasma products. Nucleic acid amplification techniques are the methods of choice for the detection of viruses, due to their high specificity and sensitivity. NAT assays are beneficial tools for the identification of contaminated mini-pools or plasma pools and the quantification of B19 contamination. They may also be valuable for testing the removal of B19 virus during manufacturing: since the virus may not be completely inactivated or removed by chemical or physical treatments, residual B19 contamination should always be checked. Solvent-detergent treatments fail to destroy B19 capsids because of the absence of a lipid-envelope, and heat treatments (pasteurization and dry-heat methods) cannot guarantee a complete viral inactivation because of the variable heat sensitivity of the virus.

Archives of Virology, Feb 1, 1995

Genomic variability in human parvovirus B 19 was analysed by direct partial sequencing of differe... more Genomic variability in human parvovirus B 19 was analysed by direct partial sequencing of different isolates collected in Italy between 1989 and 1994. DNA was purified from viremic serum samples and the region of viral genome coding for structural proteins was amplified by polymerase chain reaction and partially sequenced (nt. 2400-3400). Data were compared to reference isolates Wi (U.K., 1973) and Au (U.S.A., 1982). The average relative distance between isolates was estimated at the low level of0.61%, comparable with the distance to reference isolates. Out of 22 nucleotide substitutions found, 9 resulted in amino acid changes. From our results, this region of human parvovirus B19 genome appears to be stably conserved.

Journal of Clinical Microbiology, Jun 1, 1997

We describe a simple and rapid dot immunoperoxidase assay for the direct detection of parvovirus ... more We describe a simple and rapid dot immunoperoxidase assay for the direct detection of parvovirus B19 capsid antigens in human sera. The assay was performed with serum specimens dotted onto nylon membranes. VP1 and VP2 B19 antigens, which represent 4 and 96% of the capsid, respectively, were detected with a pool of monoclonal antibodies directed against the two proteins, and the complex was visualized by immunoperoxidase staining. The assay could be performed in about 4 h, and positive results were revealed at the end of the reaction as dark blue spots on the nylon membrane at the site of positive specimens. A total of 541 serum samples from different subjects and with different laboratory evaluations with regard to B19 infection were analyzed. The results obtained by the dot immunoperoxidase assay were compared with the results obtained for the presence of B19 DNA by dot blot hybridization and nested PCR. With optimized working conditions, the dot immunoperoxidase assay was able to detect the presence of B19 with a sensitivity comparable or slightly higher than that achieved by dot blot hybridization but less than that achieved by nested PCR. Since the level of sensitivity of the dot immunoperoxidase assay proved to be appropriate for the detection of acute B19 infection, and since the cost, time to a result, and versatility of the assay are important issues, from our evaluation, the dot immunoperoxidase assay described may be particularly suitable for large-scale screening of samples and a good alternative to DNA detection methods in the routine laboratory evaluation of B19 infection.

Pathology, May 1, 1988

It has been shown that viruses can induce alterations in the content and distribution of cytoskel... more It has been shown that viruses can induce alterations in the content and distribution of cytoskeleton structures, particularly actin microfilaments and microtubules. An immunomorphologic study of the cytoskeleton components of various EBV-infected cell lines with expression of different functions of EBV genome has been performed using antibodies to each of its three major components. Intermediate filaments and microtubules were similarly represented in all examined lymphoblastoid cell lines. The distribution of actin microfilaments, on the contrary, differed significantly from cell line to cell line. It is concluded that the morphologic expression of actin might depend on the expression of EBV genome. Furthermore, some of these cell lines might represent a useful substrate for the identification of anticytoskeleton antibodies, mainly anti-actin antibodies, in human sera.

Virus Research, Dec 31, 1986

Virology, Nov 1, 2008

Comprehension of the pathogenetic potential of human parvovirus B19 requires the definition of th... more Comprehension of the pathogenetic potential of human parvovirus B19 requires the definition of the complete spectrum of cellular tropism and a functional analysis of the viral genome in infected cells. In this study, we carried out a systematic functional analysis of B19 virus genome in the course of infection of susceptible bone marrow mononuclear cells and myeloblastoid UT7/EpoS1 cells, in terms of dynamics of nucleic acid synthesis. A PCR array was designed and a comprehensive analysis was performed by quantitative PCR and RT-PCR, yielding extended information on the presence and abundance of the diverse classes of viral nucleic acids, on the temporal regulation of genome expression and on its relationship with the cell cycle. The analysis performed indicate that the synthesis of viral nucleic acids is correlated to the progression through the S phase of the cell cycle, that an extended pattern of transcriptional activity occurs throughout the course of infection, with a maximal rate of transcription preceding the onset of S-phase dependent replication of the viral genome, and that utilization of transcript processing signals is relatively constant throughout the course of infection. The information obtained led to the definition of a unified model of functional and expression profiling of parvovirus B19 genome.

Analytical Biochemistry, Mar 1, 1998

A chemiluminescent system has been developed for ultrasensitive, quantitative analysis as well as... more A chemiluminescent system has been developed for ultrasensitive, quantitative analysis as well as visualization of the spatial distribution of biomolecules such as antigens, enzymes, antibodies, DNA probes in tissue, or cells. The system consists of a low-light imaging Vidicon videocamera connected to an optical microscope, able to measure light at the single photon level and perform 3D image analysis of the subcellular distribution of the analyte. The concentration and the spatial distribution of enzymes, or enzyme-labeled biospecific reagents can be determined using appropriate chemiluminescent substrates. Analytes are also determined with coupled enzymatic reactions terminating in light emission. Oxirane acrylic beads (250-m-diameter macroporous particles) with immobilized horseradish peroxidase have been used as a model system to optimize the experimental conditions in terms of signal intensity and spatial resolution as a function of different chemiluminescent substrates such as luminol/enhancer/H 2 O 2 and acridancarboxylate ester/ H 2 O 2. Localization of oxirane beads immobilized acetylcholinesterase has been also used to optimize a system in which the detection and localization of the primary enzyme involves two secondary enzymes in solution, choline oxidase and horseradish peroxidase, leading to a final light emission. Immunoenzymatic reactions for the detection of viral antigens and in situ hybridization assays for the detection of viral DNAs (cytomegalovirus, herpes simplex virus) have been performed in cells using peroxidase-labeled antibodies or cDNA probes and the analytical performance of different chemiluminescent substrates for the enzyme has been evaluated. The results obtained showed the possibility to sharply image the bioprobes in single cells and peroxidase is a suitable label when luminol/ H 2 O 2 system is used in conjunction with enhancer as in the ECL and SuperSignal Ultra reagents; other substrates such as Lumigen PS-3, despite adequate detectability, showed problems of localization of the signal as a result of the relatively long half-life of the excited emitting species and its diffusion in the chemiluminescent cocktail. The system has proven to be highly sensitive, able to perform quantitative analysis, and relatively simple.

Analytical Biochemistry, Sep 15, 2004

A miniaturized polymerase chain reaction (PCR) chemiluminescent enzyme immunoassay (CLEIA) based ... more A miniaturized polymerase chain reaction (PCR) chemiluminescent enzyme immunoassay (CLEIA) based on a 384-well microtiter plate for detection and typing of oncogenic high-and low-risk human papillomavirus (HPV) in genital lesions is described. The assay relies on PCR consensus amplification, hybridization of the digoxigenin-labeled product by means of type-specific biotin-labeled oligoprobes immobilized on the streptavidin-coated wells of a 384-well microtiter plate, and quantification by means of a horseradish peroxidase-labeled antidigoxigenin antibody and chemiluminescence detection. The method provides semiquantitative information on the viral load,