New evidence of cutaneous leishmaniasis in north-eastern Italy.

BACKGROUND: Human leishmaniasis is on increase in the Mediterranean Europe. However, the exact prevalence of cutaneous leishmaniasis (CL) is largely unknown as underdiagnosis and under reporting are common. OBJECTIVE: To evaluate epidemiological, clinicopathological and microbiological aspects of CL cases occurring in the Bologna Province, north-eastern Italy. METHODS: We performed a retrospective, observational study on CL cases diagnosed in the Bologna Province between January 2013 and December 2015. RESULTS: During 2013-2015, 30 cases of CL were identified in the Bologna Province with an average incidence of 1.00/100 000, with an increase of fourfold to 12-fold as compared to previous years. 16 of 30 (53%) CL cases presented as single, typical lesions. CL diagnosis was carried out by histological and molecular techniques, although in 7 of 29 (24%) PCR-positive cases, amastigotes were not visible on histology. CONCLUSIONS: We report new evidence of CL cases in a focal area of north-eastern Italy in 2013-2015. Our study highlights the importance of CL surveillance in the Mediterranean basin and emphasizes the need for the molecular laboratory surveillance of CL in endemic areas.

Ongoing outbreak of visceral leishmaniasis in Bologna Province, Italy, November 2012 to May 2013.

An increased number of autochthonous visceral leishmaniasis (VL) cases has recently been reported in Bologna Province in northern Italy. Over six months from November 2012 to May 2013, 14 cases occurred, whereas the average number of cases per year was 2.6 (range: 0-8) in 2008 to 2012. VL was diagnosed in a median of 40 days (range: 15-120) from disease onset. This delay in diagnosis shows the need for heightened awareness of clinicians for autochthonous VL in Europe.

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.

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. DESIGN: B19 DNA detection in fetal samples and serological signs of B19 infection in the respective mothers. Samples collected between January 2000 and July 2008. SETTING: Microbiology, University of Bologna, Bologna, Italy. SAMPLES: One hundred thirty-five fetal samples (58 fetal cord blood and 77 amniotic fluid samples) and 109 serum samples collected from 109 pregnant women. METHODS: Validated and certified in situ hybridisation assay (ISH) and polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) were performed on fetal samples to detect B19 DNA. B19-specific antibodies were investigated in maternal serum samples by a commercial enzyme immunoassay. MAIN OUTCOME MEASURES: Parvovirus B19 DNA detection in fetal specimens was analysed in relation to maternal serological signs of infection. RESULTS: Parvovirus B19 DNA was detected in 22.41% of fetal cord blood and 36.36% of amniotic fluid samples. A statistically significant difference was found between DNA detection by ISH (23.70%) and PCR-ELISA (14.81%) (P= 0.004). Only 11.76% of fetuses with virological diagnosis of B19 infection were from women with serological signs of acute/recent B19 infection. CONCLUSIONS: Diagnosis of fetal parvovirus B19 infection cannot always rely on maternal serological investigations but rather on the virological analysis of fetal samples. Both fetal cord blood and amniotic fluid samples are suitable for diagnosis, but the detection of B19 DNA in the cells of amniotic fluid samples by ISH proved to be the most reliable diagnostic system.

Efficient treatment of paraffin-embedded cervical tissue for HPV DNA testing by HC-II and PCR assays.

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.

Relevance of B19 markers in serum samples for a diagnosis of parvovirus B19-correlated diseases.

In order to evaluate the optimal and essential diagnostic test(s) for a correct diagnosis of B19 diseases, 344 consecutive serum samples were tested from 344 patients with clinical suspicion of B19 infection during an epidemic period (early Spring-Autumn 2000). Sera were tested for B19 DNA by a standardized competitive polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) and dot-blot hybridization and for specific IgM and IgG by ELISA. Of 344 patients examined, 125 were positive for markers of B19-associated disease: 49 had both B19 DNA and IgM, 50 had B19 DNA without IgM, and 26 had IgM without B19 DNA. After examination of the different patterns of B19 markers as diagnostic tools for B19 infection, IgM determination detected only 60% of B19-documented infections. IgM tests were nevertheless fundamental, as they were the unique diagnostic marker in 20.8% of documented infections (26 of 125 patients), in the diagnosis of recent, but still symptomatic infections when B19 DNA was no longer detectable. The determination of B19 DNA with PCR permitted detection of 79.2% of infections and therefore represented an essential test. PCR was fundamental for the diagnosis of B19 disease, as the unique diagnostic marker in 32% of documented infections (50 of 125 patients), both in acute infections at the onset of symptoms before the appearance of immunological response, and during the course of persistent B19 infections in which IgM had cleared. The contemporaneous determination of B19 DNA by PCR and specific IgM appears to be the most appropriate diagnostic protocol for the correct laboratory diagnosis of B19 infection.

Self-assembling poly(vinyl alcohol) derivatives, interactions with drugs and control of release.

The self-assembling properties of poly(vinyl alcohol) substituted with 2-hydroxypropyltrimethylammonium and with acyl chains of different molecular weights (butyryl, capryloyl, lauroyl, or myristoyl) were evaluated to assess the conditions favoring interaction with a poorly soluble drug such as indomethacin to increase its availability. To evaluate the effect of drug-polymer interactions on the solubility of the drug, phase-solubility diagrams were obtained from each substituted polymer at pH 2.0, 5.5, and 7.4 in the presence of indomethacin. To evaluate the availability of the free drug in solution, release profiles of the free drug from drug-polymer physical mixtures were obtained by a dissolution-diffusion apparatus containing a dialysis membrane allowing diffusion of the free drug towards a receiving phase where its concentration was determined over time. The phase-solubility diagrams revealed increasing drug solubility on increasing the polymer concentration. The drug-polymer affinity was slightly increased by lengthening the chain of the substituent on the polymer and was strongly increased by raising the pH of the aqueous phase. The thermodynamic evaluation of the drug-polymer interactions indicated that the interaction is enthalpically driven while the increase in drug-polymer affinity with increasing chain length could be attributed to an entropic contribution. The free drug availability from the drug-polymer systems increased on enhancing the drug-polymer affinity because it corresponded to an increase in the solubilizing effect of the polymer on the drug.

Detection of adeno-associated virus DNA in female genital samples by PCR-ELISA.

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 specificity and the sensitivity of conventional PCR with an objective interpretation of the results. In the PCR-ELISA, a defined number of cells from cervical specimens were digested and amplified with concomitant digoxigenin labeling. Digoxigenin-labeled amplified products hybridized to a specific 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% specific. 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.

Identification of an immunodominant peptide in the parvovirus B19 VP1 unique region able to elicit a long-lasting immune response in humans.

The immune response against parvovirus B19 is mainly directed against the two structural proteins, VP1 and VP2. The amino terminal half of the VP1 unique region has been shown to elicit a dominant immune response in humans, more effective than other linear epitopes and also it has been seen to contain significant neutralizing linear epitopes. Three overlapping recombinant peptides corresponding to amino acids 2-40 (VP1-A), amino acids 32-71 (VP1-B), and amino acids 60-100 (VP1-C) of the VP1 unique region were produced by a procaryotic expression system. These peptides were used as antigens in a Western blot assay to detect specific immunoglobulin G (IgG) in serum samples from blood donors of different age groups with documented signs of a past B19 infection. Fragment VP1-C appeared significantly immunodominant over the other peptides, reacting with specific IgG in 86% of serum samples. The fragment VP1-C corresponds to a sequence with a known neutralizing activity and seems able to elicit a long-lasting immune response because specific IgG were present in blood donors of all age groups. VP1-C would therefore appear to be an attractive candidate as a component of a subunit vaccine.

Differential IgM response to conformational and linear epitopes of parvovirus B19 VP1 and VP2 structural proteins.

The IgM immune response against conformational and linear epitopes of B19 structural proteins VP1 and VP2 was examined in serum samples with a suspect B19 infection to determine the most suitable antigen for use in IgM detection and also to evaluate a possible relationship between the course of B19 infection and the presence of epitope type-specific IgM. The detection of IgM against conformational epitopes was performed by ELISA using undenatured VP1 and VP2 antigens whereas the detection of IgM against linear epitopes was performed by Western blot assays using denatured VP1 and VP2. IgM immune response against VP1 conformational epitopes appeared dominant, being detected in all serum samples positive for specific IgM, whereas IgM against VP2 linear antigen were found less frequently, being identified in less than half of the B19 IgM positive sera. In the examination of the course of infection, IgM against VP1 conformational epitopes appeared in the active phase of B19 infection at the same time and with the same frequency as IgM anti VP2 conformational epitopes and anti linear VP1 epitopes. IgM against VP1 conformational epitopes were seen to be long-lasting because in the recent phase of infection they were still present when other specific IgM were absent. During the active phase of B19 infection, IgM against VP2 linear epitopes were less frequently found than other specific IgM and in the recent phase they underwent a rapid temporal diminution. The data demonstrate that a sensitive B19 IgM test needs to be performed in diagnostic laboratories by ELISA using conformational B19 antigens; Western blot assays can be used only as confirmatory tests using VP1 linear antigens.

A system to enhance the sensitivity of digoxigenin-labelled probe: detection of B19 DNA in serum samples.

A highly sensitive dot-blot hybridisation assay for the routine screening of numerous samples is described, using parvovirus B19 as a model. Digoxigenin-labelled B19 DNA probe was constructed by PCR, hybrids were detected by an anti-digoxigenin monoclonal antibody followed by a second step, using anti-mouse antibodies conjugated to an alkaline phosphatase-dextran complex (EnVision, Dako) was carried out. The sensitivity of the assay was evaluated using both colourimetric and chemiluminescent substrates for the alkaline phosphatase and was compared with a dot-blot hybridisation assay using the digoxigenin-labelled probe and a standard detection system. With the colourimetric substrate, the EnVision system was able to detect 10 fg of B19 DNA, while with the chemiluminescent substrate the sensitivity increased by up to 2 fg (6 x 10(2) genome copies). This detection system was shown to increase the sensitivity of the assay compared to the standard colourimetric visualisation for the digoxigenin-labelled probe, which could detect 0.1 pg. On account of its sensitivity and specificity the dot-blot hybridisation assay together with the chemiluminescent substrate for the EnVision detection system was used to analyse 760 serum samples; the same sera were tested for B19 DNA with the standard colourimetric visualisation for the digoxigenin-labelled probe used routinely in the diagnostic laboratory.

Synthesis and antiviral assays of some benzimidazole nucleosides and acyclonucleosides.

Some benzimidazole nucleosides and acyclonucleosides were synthesized and tested in vitro as antiviral agents. None of them showed significant activity. Replacement of the benzenesulphonyl group at N-1 with the ribofuranosyl moiety or with the acyclovir side-chain was deleterious.

Tyramide signal amplification of biotinylated probe in dot-blot hybridization assay for the detection of parvovirus B19 DNA in serum samples.

Highly sensitive assay systems are necessary for large-scale virological screenings. We evaluated the use of tyramide signal amplification (TSA) for biotinylated probe in dot-blot hybridization assay to detect B19 DNA in serum samples. The probe was constructed by PCR and directly labeled with biotin during amplification reaction. The sensitivity of the dot-blot hybridization assay with TSA detection method was evaluated in comparison with a hybridization assay using the direct detection of biotinylated probe by streptavidin-biotin-alkaline phosphatase substrate. The TSA detection was able to detect 1 pg of B19 DNA and proved to be 10-50 times more sensitive than the hybridization assay with the direct detection of biotinylated probe. The analysis of 720 serum samples by TSA detection of biotinylated probe showed that the assay may be a valid diagnostic tool in routine testing of B19 DNA in serum samples.

Synthesis and antiviral assays of some 2-substituted benzimidazole-N-carbamates.

Some 2-substituted benzimidazole-N-carbamates were synthesized and tested in vitro for antiviral activity. Two derivatives were active at noncytotoxic concentrations. The results confirmed the importance of the substituents at the 2-position of benzimidazole; an isopropylcarboxamide group led to the best activity.

Chemiluminescence Western blot assay for the detection of immunity against parvovirus B19 VP1 and VP2 linear epitopes using a videocamera based luminograph.

A chemiluminescent Western blot (WB) assay was developed to detect the immune status against linear epitopes of parvovirus B19 structural proteins VP1 and VP2. The chemiluminescent WB assay combined the sensitivity of chemiluminescent substrates and the objective evaluation of the luminescent signal obtained with a new system which consists of a videocamera-based, high-performance, low light level imaging luminograph connected to a personal computer for image analysis. The potential for diagnostic purposes was evaluated using reference serum samples and 75 clinical samples from patients with different clinical conditions and laboratory evaluations regarding B19 infection. The chemiluminescent Western blot assay provided reproducible results, an objective evaluation of the results and a semi-quantitative analysis of the presence of antibodies against VP1 and VP2 in human sera. The chemiluminescent Western blot assay proved more sensitive than the classic colourimetric Western blot assay.

Standardization of a PCR-ELISA in serum samples: diagnosis of active parvovirus B19 infection.

To standardize a PCR assay for the detection of parvovirus B19 DNA in serum samples three different sample treatments were evaluated on the basis of the efficiency of recovery, reproducibility, convenience of sample handling, and presence of PCR inhibitors. Moreover, the presence of an internal standard competitor as the working reagent at one defined concentration in a competitive PCR-ELISA has been suggested as a valid tool to standardize and validate the assay. The results indicated that serum sample treatment by rapid heating fulfilled the criteria for a routine practice in the diagnostic laboratory. Titration experiments carried out to define the optimal amount of the internal standard competitor to use in PCR-ELISA showed that at 2 x10(2) competitor copies, any amplification interferences between target and competitor sequences were avoided. The internal standard competitor in a competitive PCR-ELISA allows the detection of false-negative results due to PCR inhibitors in the samples or large amounts of target DNA. Heating treatment and competitive PCR-ELISA for the detection of parvovirus B19 DNA were applied to the testing of 347 serum samples, which were submitted to the laboratory for B19 investigation. Of the 34 serum samples that were positive for B19 DNA, 15 were from adult patients and 19 from pediatric subjects. B19 infection was associated with haematological disorders, nonimmunological foetal hydrops, atypical rash, arthropathies, hepatic dysfunction, nonspecific symptoms, and congenital infections.

Prenatal diagnosis of parvovirus B19-induced hydrops fetalis by chemiluminescence in situ hybridization.

Parvovirus B19 can be transmitted transplacentally from the infected mother to the fetus during pregnancy, and hydrops fetalis, abortion, or stillbirth can result. In our study we explored the use of chemiluminescence in situ hybridization to detect B19 DNA on cord blood cells, amniotic fluid cells, and pleuric fluid cells from several cases of hydrops fetalis. B19 DNA was detected by using digoxigenin-labeled probes immunoenzymatically visualized with the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal emitted from the hybridized probes was detected, analyzed, and measured with a high-performance, low-light-level imaging luminograph connected to an optical microscope and to a personal computer for the quantification and localization of the chemiluminescent emission inside individual cells.

IgG immune response to B19 parvovirus VP1 and VP2 linear epitopes by immunoblot assay.

Human B19 parvovirus recombinant capsid proteins VP1 and VP2 were expressed in E. coli and purified. Recombinant proteins were used to detect a specific IgG immune response against VP1 and VP2 linear epitopes by immunoblot assay. A total of 222 serum samples from 218 apparently immunocompetent subjects with different clinical conditions and laboratory evaluations with regards to B19 infection were analyzed. The sera had previously been tested for B19 DNA and for specific IgM and IgG against VP2 conformational antigens by ELISA assay. The data show that, during the active or very recent phase of infection, IgG anti-VP1 linear epitopes appear in concomitance and with the same frequency as IgG anti-VP2 conformational antigens. IgG against conformational VP2 antigens and against linear VP1 epitopes seem to persist for months or years in the majority of individuals. IgG against VP2 linear epitopes are generally present during the active or very recent phase of infection and during the convalescent phase, while they are present only in about 20% of subjects with signs of a past B19 infection.

IgG response to the immunoreactive region of parvovirus B19 nonstructural protein by immunoblot assay with a recombinant antigen.

A total of 190 serum samples from patients with different clinical manifestations in whom B19 parvovirus infection was suspected and 108 control serum samples were analyzed for the IgG response against parvovirus B19 nonstructural protein. An immunoblot assay was developed using the immunoreactive region of the B19 nonstructural protein as recombinant antigen. Virologic (B19 DNA) and serologic (IgM and IgG anti-VP) markers of B19 infection were also investigated. There was an even distribution of IgG nonstructural antibodies in serum samples of parvovirus B19-infected patients and controls (27.7% and 21.7%, respectively) that were mainly associated with patterns of past B19 infection.

Intra-uterine parvovirus B19 infection and meconium peritonitis.

B19 fetal infection has been associated with hydrops or fetal death. We report four cases of meconium peritonitis in hydropic fetuses with laboratory diagnosis of B19 infection. Parvovirus B19 DNA was detected by in situ hybridization both in cord blood and in amniotic cells in three fetuses, while in one case only cord blood was available and proved positive. Signs of active or recent B19 infection in maternal serum samples were documented only in two cases, which proved positive for specific IgM antibodies anti-B19. Maternal B19 infections were asymptomatic and fetal anomalies were observed during a routine ultrasound scan. A common feature of the hydropic fetuses was the presence of abdominal ascites concomitant with or preceding alterations, suggesting meconium peritonitis. The four pregnancies had a preterm outcome: in two cases infants recovered following surgical treatment, in one case spontaneously, and the other one was stillborn. Since vascular inflammation has been documented in B19 infection and congenital bowel obstruction results from vascular damage during fetal life, our observation suggests the need for investigating B19 infection in the presence of meconium peritonitis for a better understanding of the pathogenetic potential of B19 parvovirus in intra-uterine infection.