Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro.

The nucleolus is a multifunctional nuclear compartment widely known to be involved in several cellular processes, including mRNA maturation and shuttling to cytoplasmic sites, control of the cell cycle, cell proliferation, and apoptosis; thus, it is logical that many viruses, including herpesvirus, target the nucleolus in order to exploit at least one of the above-mentioned functions. Recent studies from our group demonstrated the early accumulation of the incoming ppUL83 (pp65), the major tegument protein of human cytomegalovirus (HCMV), in the nucleolus. The obtained results also suggested that a functional relationship might exist between the nucleolar localization of pp65, rRNA synthesis, and the development of the lytic program of viral gene expression. Here we present new data which support the hypothesis of a potentially relevant role of HCMV pp65 and its nucleolar localization for the control of the cell cycle by HCMV (arrest of cell proliferation in G1-G1/S), and for the promotion of viral infection. We demonstrated that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phase, its nucleolar accumulation is prominent in G1 and G1/S, but very poor in S or G2/M. This correlates with the observation that only cells in G1 and G1/S support an efficient development of the HCMV lytic cycle. We propose that HCMV pp65 might be involved in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. Co-immunoprecipitation experiments have permitted to identify nucleolin as one of the nucleolar partners of pp65.

Clinical and molecular observations of two fatal cases of rotavirus-associated enteritis in children in Italy.

Two fatal cases of infantile rotavirus enteritis occurred in northern Italy in 2005. Both children were severely dehydrated, and death was related to severe cerebral edema. Histological examination demonstrated extensive damage of the intestinal epithelium, villous atrophy or blunting, and macrophage infiltration. The two rotavirus strains were of the G1P[8] type and the long electropherotype. The 2005 G1P[8] rotaviruses differed in the NSP4, VP3, VP4, and VP7 genes from G1P[8] rotaviruses circulating in 2004, suggesting the onset of a new G1P[8] strain in the local population.

Case report: detection of rotavirus RNA in the cerebrospinal fluid of a child with rotavirus gastroenteritis and meningism.

Although case reports have described detection of rotavirus (RV) in extraintestinal sites such as the liver, kidney, and central nervous system (CNS) of children with RV gastroenteritis, CNS localization in RV infection seems to be rare. RT-PCR and nucleotide sequencing detected a G1P[8] strain in the stool and cerebrospinal fluid (CSF) samples of a patient with concurrent RV-associated enteritis and CNS signs. Upon sequence analysis, the viruses detected in the CSF was identical to the virus detected in the stools. In the VP7- and VP4-based phylogenetic dendograms the strain clustered within the G1-Ic sub-lineage and the P[8]-III lineage. This study supports the hypothesis that RV infection was able to spread from the intestinal tract to the CNS, and likely played a role in the onset of neurological disease.

Pancreatic pseudocyst-inferior vena cava fistula causing caval stenosis, left renal vein thrombosis, subcutaneous fat necrosis, arthritis and dysfibrinogenemia.

AIM: We describe the case of a 38 year old man, with a story of alcohol abuse, who developed a very painful nodular subcutaneous fat necrosis, fever and polyarthritis, denying any abdominal symptoms due to a pancreatic pseudocyst-inferior vena cava fistula. MATERIAL OF STUDY: The authors discuss the unusual and protracted course with intermittent hyperamylasemia and hyperlipasemia related to clinical manifestations such as subcutaneous fat necrosis, polyarthritis, pleural effusion and dysfibrinogenemia, and vascular complications as inferior vena cava stenosis and left renal vein thrombosis without abdominal symptomatology. RESULTS: After ultrasonograms and CT Scans showing a 3-4 cm cyst at the pancreatic head with a solid bud protruding into the pseudocystic cavity, and an ERCP showing a communication between the pancreatic duct and the pseudocyst but failing in demonstrating the vascular fistula, the patient underwent a Roux-en-y pseudocyst-jejunostomy and suture of the caval communication leading to complete recovery with normalization of laboratory findings. DISCUSSION: In our case, the locally sclerosing activity of the enzymes in the endothelium led to a communication between the inferior vena cava and the pseudocyst and to a complete thrombosis of the left renal vein and to a stenosis of the inferior vena cava itself The fluctuance of the symptomatology severity was probably due to an intermittent opening of the passage between pseudocyst and vena cava. Such a clinical case, to the author knowledge, has never been reported. CONCLUSION: When in presence of very high levels of amylasemia and lipasemia in spite of the paucity of abdominal symptomatology, and the onset of unusual complications such as panniculitis, pleural effusion, arthritis and coagulative disorders, a pancreatic pseudocyst-inferior vena cava fistula should be kept in consideration during diagnosis.

Modulatory effect of rRNA synthesis and ppUL83 nucleolar compartmentalization on human cytomegalovirus gene expression in vitro.

The nucleolus is a nuclear domain involved in the biogenesis of ribosomes, as well as in many other important cellular regulatory activities, such as cell cycle control and mRNA processing. Many viruses, including herpesviruses, are known to exploit the nucleolar compartment during their replication cycle. In a previous study, we demonstrated the preferential targeting and accumulation of the human cytomegalovirus (HCMV) UL83 phosphoprotein (pp65) to the nucleolar compartment and, in particular, to the nucleolar matrix of lytically infected fibroblasts; such targeting was already evident at very early times after infection. Here we have investigated the possible effects of rRNA synthesis inhibition upon the development of HCMV lytic infection, by using either actinomycin D or cisplatin at low concentrations, that are known to selectively inhibit RNA polymerase I activity, whilst leaving RNA polymerase II function unaffected. Following the inhibition of rRNA synthesis by either of the agents used, we observed a significant redistribution of nucleolar proteins within the nucleoplasm and a simultaneous depletion of viral pp65 from the nucleolus; this effect was highly evident in both unextracted cells and in nuclear matrices in situ. Of particular interest, even a brief suppression of rRNA synthesis resulted in a very strong inhibition of the progression of HCMV infection, as was concluded from the absence of accumulation of HCMV major immediate-early proteins within the nucleus of infected cells. These data suggest that a functional relationship might exist between rRNA synthesis, pp65 localization to the nucleolar matrix and the normal development of HCMV lytic infection.

Molecular characterization of group C rotaviruses detected in children in Italy.

BACKGROUND: Group C rotavirus (GCRV) infection has been described worldwide in children and adults either as sporadic cases or large outbreaks of gastroenteritis but GCRV epidemiology is still unclear. OBJECTIVE: To acquire molecular information on GCRV infection in children hospitalized with gastroenteritis in Italy. STUDY DESIGN: Stools positive for rotavirus-like-particles by electron microscopy during the 2004-2005 surveillance for rotavirus infection in Parma, Italy, were screened by polyacrylamide gel electrophoresis. GCRV strains detected were characterized by sequence and phylogenetic analysis of the genes encoding the capsid proteins VP4, VP6 and VP7. RESULTS: Two of 856 samples (0.23%; 0.7% of 273 samples containing rotavirus-like particles) contained GCRV. These Italian strains were virtually identical in the 3 genes and were closely related to human strains identified in Asia, rather than to strains of European origin. CONCLUSIONS: These results confirm the circulation of GCRVs in Europe and demonstrate genetic heterogeneity among European GCRVs. Inclusion of GCRVs in the diagnostic algorithms for childhood diarrhoea could be helpful in monitoring temporal changes in the GCRV epidemiology, likely under the influence of the changing demographic dynamics.

Primary appendiceal tumors: report on 10 cases.

We report our experience on 10 patients with primary tumors of the appendix treated at our institution from 1998 to 2005. There were 5 women and 5 men, with a mean age of 59.1 years. Laparotomy was performed in 4 cases; whereas, the other 6 patients underwent laparoscopic exploration: Three operations were completed laparoscopically, and 3 were converted to laparotomy. Six tumors were malignant, and the remaining were benign. Proportions of perioperative and late mortality were both 10%. Two of the four patients with benign tumors died from causes unrelated to the appendiceal neoplasm. The 6 patients with malignant tumors and the other 2 with benign disease were alive and disease free after a mean follow-up of 43 months. Despite the rarity of appendiceal primary tumors, surgeons should be aware of these neoplasms for making correct treatment decisions. We stress the importance of laparoscopic exploration in the management of appendiceal masses.

Evaluation of rubella virus immunoglobulin G (IgG) and IgM assays with the new Vidia instrument.

For rubella virus immunoglobulin G (IgG) and IgM detection, Vidia assays were compared to Vidas, AxSYM, and Liaison assays with 419 serum samples. Only Vidia produced a sensitivity of 100% for IgG and IgM. Vidia specificities were 98.4% for IgG and 99.8% for IgM, versus Vidas specificities of 100 and 99.3%. Vidia IgG and IgM assays performed equally well.

Molecular characterization of VP4, VP6 and VP7 genes of a rare G8P[14] rotavirus strain detected in an infant with gastroenteritis in Italy.

In this study, the molecular characterization of a rare G8P[14] group A rotavirus (GARV) strain detected in Northern Italy during the 2004-2005 epidemiological rotavirus season is described. Two hundred and seventy three rotavirus-like particle positive stools out of 856 stools from children (31.9%) hospitalized with gastroenteritis were analyzed using polyacrilamide gel electrophoresis and 271 GARVs were genotyped by VP7 and VP4 specific RT-PCRs. One strain (PR/1300/04) with a long electropherotype (e-type) displayed the G8 specificity and was VP4 un-typeable. The P and the subgroup (SG) specificities were determined by sequencing the VP4 and the VP6 gene, respectively. The PR/1300/04 strain exhibited P[14] and SGI specificities. By sequence and phylogenetic analyses of the VP4, VP6 and VP7 amplicons, the PR/1300/04 VP4 and VP6 genes were demonstrated to be of human rotavirus origin, with the VP4 gene closely related to the human Italian PA169 strain (G6P[14]), while the VP7 gene was of animal origin (bovine). These data suggest that the Italian PR/1300/04 strain could be a reassortant between a PA169-like Italian strain with P[14] specificity, long e-type and SGI, and a G8 animal strain. The increasing number of reports of atypical GARVs in humans suggests that interspecies transmission of genes greatly contributes to the GARV genetic evolution.

An 8-year survey on the occurrence of imported malaria in a nonendemic area by microscopy and molecular assays.

Our study aimed to describe the occurrence of imported malaria in a nonendemic area (Parma, Italy) during the period 2000 to 2007, comparing the data obtained by microscopy and molecular assays targeting plasmodial 18S subunit rRNA gene. The prevalence of imported malaria in Parma was 21.8% by microscopy and 22.7% by polymerase chain reaction (PCR). Plasmodium falciparum accounted for 81.1% of the cases, followed by Plasmodium ovale (8.8%), Plasmodium vivax (3.8%), and Plasmodium malariae (1.9%). Mixed infections accounted for 4.4% of the cases. In this study, PCRs proved to be more sensitive and specific than microscopy and changed the picture of malaria epidemiology in Parma, detecting additional cases of malaria undiagnosed by microscopy and allowing speciation of plasmodia in cases misidentified by microscopy. Generally, imported malaria cases reflect the number of immigrants who visit their native countries, in particular, West Africa, explaining the increased prevalence of P. ovale cases among non-P. falciparum infections in Parma.

Myiasis of the scalp due to Dermatobia hominis in a traveler returning from Brazil.

This article describes a case of myiasis by Dermatobia hominis diagnosed in a young Italian man returning from a vacation through Brazil. Considering the increasing number of travels to tropical and subtropical areas, clinicians in nonendemic areas must think about the possibility of imported unusual infestations during their daily practice.

Occurrence of human intestinal spirochetosis in comparison with infections by other enteropathogenic agents in an area of the Northern Italy.

The aim of this study was to investigate the occurrence of human intestinal spirochetosis (IS) by a 16S rRNA restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) in a selected group (234) of patients with gastrointestinal complaints and/or potential risk factors for IS in comparison with the occurrence of infections by other enteropathogenic agents. By using 16S rRNA RFLP-PCR, 16 patients (6.8%) with IS were found (11 infected by Brachyspira aalborgi, 3 by Brachyspira pilosicoli, and 2 by both species); moreover, 10 patients with gastroenteric viruses (4.2%), 13 with enteropathogenic bacteria other than intestinal spirochetes (5.5%), and 24 with intestinal parasites (10.2%) were found. This study provides an enhancement of the knowledge about the distribution of IS, suggesting that it may be more frequent than suspected and that clinicians should consider IS when patients present with long-standing diarrhea. Moreover, 16S rRNA RFLP-PCR might be a powerful tool not only for diagnostic purpose but also to investigate the occurrence of IS just on fecal samples, not requiring invasive diagnostic techniques.

Human intestinal spirochaetosis in Parma: a focus on a selected population during 2002-2005.

BACKGROUND AND AIM OF THE WORK: Human intestinal spirochaetosis (HIS) is a large bowel infection characterised by the colonization of the intestinal mucosa by spirochaetes belonging to the genus Brachyspira. The causative agents of HIS are Brachyspira aalborgi and Brachyspirapilosicoli. Symptoms of the infection, even if not specific, are long standing diarrhoea, abdominal pain, meteorism and rectal bleeding and sometimes they can suggest the clinical suspect of inflammatory bowel diseases or rectal carcinoma. Since poor data were available on the prevalence of this infection, the aim of our study was to describe the occurrence of this infection in our area in the period 2002-2005. METHODS: During a period of 4 years we analysed 297 faecal samples from 99 patients selected by potential risk factors and symptomatology suspected for HIS. The diagnosis of HIS was performed by isolation and a molecular assay based on 16S rDNA restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR). RESULTS: From 2002 to 2005 we detected 12 cases of intestinal spirochaetosis, 7 caused by Brachyspira aalborgi, 4 by Brachyspirapilosicoli and one by both spirochaetes, which represented the first case of a mixed infection by 2 intestinal spirochaetes in our area. CONCLUSIONS: Despite the fact that HIS seems to be a low prevalence infection in our area, in a strongly selected population we found 12 cases of this infection (12.12%). These results stimulate us to extend the research of intestinal spirochaetosis in the general population, when long standing gastrointestinal disorders and potential risk factors are present.

Prevalence of imported malaria in Parma during 2005-2006.

BACKGROUND AND AIM OF THE WORK: Malaria is a protozoan infection caused by parasites of the genus Plasmodium (P. falciparum, P. ovale, P. vivax, P. malariae) that is transmitted from one human to another by female Anopheles mosquitoes. It can be considered a reemerging imported disease in our area because of increasing of movements from endemic countries, and nowadays it is the most common imported infection in Italy. This study describes the occurrence of imported malaria in our area between January 2005 and May 2006. METHODS: During 17 months we analysed 170 blood samples belonging to 139 patients (95 foreigners and 44 Italians) with the clinical suspect of malaria. Samples were used to prepare orange acridine and Giemsa stained thin blood films for microscopic observation and to perform an immunochromatographic assay for the detection of specific plasmodia antigens. Molecular assays (nested-PCR and Real-time PCR) were also performed in order to confirm the diagnosis. RESULTS: Thirty-six cases of malaria were diagnosed: 35 in foreigners coming from Africa and only one in an Italian who lived in Chad. Thirty-three patients were infected by P. falciparum, 1 by P. ovale, 1 by P. vivax, and a mixed infection by P. falciparum, P. ovale and P. malariae was also found. CONCLUSIONS: Malaria is usually associated with travels within areas where the infection is endemic and our data demonstrated that imported malaria in our area has a prevalence of 25.89%.

Entamoeba histolytica and Entamoeba dispar: comparison of two PCR assays for diagnosis in a non-endemic setting.

Detection of Entamoeba histolytica, the causative agent of amoebiasis, is an important goal of the clinical parasitology laboratory. The identification of Entamoeba dispar as a morphologically identical but non-pathogenic species has highlighted the need for non-microscopic detection methods able to differentiate between the two organisms. In this study we evaluated the utility of conventional PCR and real-time PCR as methods for identification and differentiation of E. histolytica and E. dispar. The second aim of this study was to determine the relative proportions of infections caused by E. histolytica and the non-pathogenic E. dispar, allowing a picture of the epidemiological situation in a non-endemic setting to be obtained. One hundred and sixty-six clinical samples (faecal and liver abscess samples and one intestinal biopsy) belonging to 108 patients were analysed. More patients with E. dispar infection (8.3%) than patients with E. histolytica infection (5.6%) were found by both PCR assays. It is concluded that routine diagnosis of invasive amoebiasis performed by a combination of microscopy, culture and serology should be complemented with a PCR assay such as real-time PCR that offers a practical and clinically acceptable alternative for rapid and accurate diagnosis of amoebic infection in patients presenting with symptoms indicative of this disease.

HBV genotypes and antiviral-resistant variants in HBV infected subjects in Northern Italy.

HBV genotypes were investigated in sera/plasma from 97 HBV positive subjects. Genotype D was revealed in 80.4% followed by E in 6.2%. Genotypes A, B, and C were also found, as well as for the first time a new combination of HBV D and G genotypes. In a cohort of subjects of this population, the relationship with lamivudine and/or famciclovir-resistant HBV mutants was also investigated. Among 12 untreated subjects, 25% carried HBV drug-resistant strains suggesting that drug-resistant variants naturally exist in untreated Italian HBV chronically infected subjects.

Prevalence of intestinal parasites in the area of Parma during the year 2005.

BACKGROUND AND AIM OF THE WORK: Intestinal parasitosis represent a relevant clinical problem, especially in developing countries, where they are responsible for morbidity and mortality in adults and children and many epidemiological data are available for these areas. The actual situation of intestinal parasitosis in Europe is not yet well investigated since they are usually not notified. We describe the occurrence of intestinal parasitosis in our laboratory from January to December 2005. METHODS: We considered all patients (1117) whose stool samples were sent to our laboratory with the suspect of intestinal parasitosis during the year 2005. Each specimen was subjected to macroscopic and microscopic examination to demonstrate the presence of worm eggs, larvae, protozoan trophozoites or cysts and to an immunochromatographic assay to detect Giardia intestinalis and Cryptosporidium spp. specific antigens. Cultures for protozoa and helminths were carried out and a PCR specific for Entamoeba histolytica/Entamoeba dispar was also performed. RESULTS: Our results indicated that 148 patients (13.24%) were affected by intestinal parasitosis. Among the 951 Italians, 96 (10%) were infected, while out of a total of 166 foreigners 52 had intestinal parasitosis (31%). Moreover, we found that 113 infections were caused by only one parasite while 35 were mixed infections. CONCLUSIONS: Intestinal parasitosis represent a remarkable cause of gastrointestinal disease and our study demonstrates that these infections are quite common in our area, affecting both Italians and non European citizens from developing countries.

Comparison between two real-time PCR assays and a nested-PCR for the detection of Toxoplasma gondii.

BACKGROUND AND AIM OF THE WORK: In recent years, the diagnosis of toxoplasmosis has been improved by Real-time PCR assays. In this study we compared the performances of two Real-time PCRs (FRET and TaqMan protocols) already described in the literature, and one nested-PCR, currently used in our laboratory for the molecular diagnosis of toxoplasmosis. METHODS: We evaluated the sensitivity and the specificity of a FRET- and a TaqMan-based Real-time PCRs targeting a 529 bp repeat region and the 18S RNA gene, respectively, and a nested-PCR, targeting the B1-gene of Toxoplasma gondii. We also tested, through nested-PCR, 46 biological samples obtained during a period of 29 months from pregnant women or immunocompromised patients with suspected T. gondii infection. RESULTS: The analytical sensitivity of nested and TaqMan PCRs was approximately 10(3) tachyzoites/ml. FRET assay showed a sensitivity of 102 tachyzoites/ml. Three out of 46 biological samples were nested-PCR-positive and these results were also confirmed by both Real-time PCRs. CONCLUSIONS: Nested- and real-time PCRs evaluated in this study resulted very sensitive and specific; in particular FRET PCR resulted more sensitive than the other assays, probably because of the greater copy number of the target sequence. Real-time PCR assays are easy-to-use, producing results faster than conventional PCR systems and reducing contamination risks.

Electron microscopy as a reliable tool for rapid and conventional detection of enteric viral agents: a five-year experience report.

BACKGROUND AND AIM OF THE WORK: Since the introduction of the electron microscope and its subsequent development, virology has made a great step forward by the improvement of the basic knowledge on viral structure, as well as by broad application of electron microscopy (EM) to viral diagnosis. In this report, we describe a five-year experience in the use of EM for the diagnosis of enteric viral infections. METHODS: Three thousand four hundred and ninety stool specimens were analyzed at the Virology Unit (Section of Microbiology, Department of Pathology and Laboratory Medicine, University of Parma, Italy) during a five-year period, from January 1999 to January 2004. The faecal extracts were subjected to EM after negative staining and were simultaneously cultured to evidence the presence of cytopathogenic agents. RESULTS: EM directly applied to the above specimens allowed the detection of several enteric viral agents, particularly evidencing those normally hard to cultivate (thus easily lost with culture methods). It also enabled diagnosis of dual gut infections, such as those from rotavirus and calicivirus. On the other hand, EM-based identification of viral agents after cell culture and ultracentrifugation of cytopathogenic agent-containing cellular extracts, allowed the identification of cultivable agents, such as picornaviruses, which can escape the direct EM detection if low concentrated. CONCLUSIONS: A rationalized use of EM on selected samples, such as stool, appears suitable in epidemiological or clinical conditions when a very rapid diagnosis is required to save time, including cases of suspected emerging viral infections.

Differentiation of leptospires of the serogroup Pomona by monoclonal antibodies, pulsed-field gel electrophoresis and arbitrarily primed polymerase chain reaction.

All reference strains described as representing separate serovars belonging to the serogroup Pomona and a clinical leptospiral isolate (LP2) from this serogroup were analyzed using a battery of 9 monoclonal antibodies, pulsed-field gel electrophoresis (PFGE) and arbitrarily primed polymerase chain reaction (AP-PCR). Monoclonal antibody analysis provided taxonomic results which were in agreement with the current classification of the serogroup Pomona into six serovars and allowed the classification of the isolate LP2 in the serovar pomona. PFGE and AP-PCR, although in general agreement with monoclonal antibody analysis, also were able to demonstrate some differences in the restriction patterns of strains Pomona, Monjakov and CB. These results indicate that these strains, grouped within serovar pomona after the introduction of bacterial restriction endonuclease analysis as the typing method, but formerly described as representing separate serovars (pomona, monjakov and cornelli, respectively), are similar but not identical to one another. This was also the case with strains 5621, the serovar mozdok reference strain, and K1, formerly described as serovar dania reference strain, but currently recognized to be a mozdok-like strain. These findings suggest that the deletion of some serovars within the serogroup Pomona, namely mozdok, cornelli, and dania, should be reconsidered. Thus, PFGE appears to be a useful tool for the serovar identification of leptospires belonging to the serogroup Pomona and for shedding light on the problem of their classification.