Elisa Franchin

Isolated Hepatocyte Transplantation for Crigler-Najjar Syndrome Type 1:

Crigler-Najjar syndrome type 1 (CN1) is an inherited disorder characterized by the absence of hepatic uridine diphosphoglucuronate glucuronosyltransferase (UDPGT), the enzyme responsible for the conjugation and excretion of bilirubin. We performed allogenic hepatocyte transplantation (AHT) in a child with CN1, aiming to improve bilirubin glucuronidation in this condition. A 9-year-old boy with CN1 was prepared with plasmapheresis and immunosuppression with prednisolone and tacrolimus. When a graft was made available, 7.5 × 109 hepatocytes were isolated and infused into the portal vein percutaneously. After 2 weeks phenobarbitone was added to promote the enzymatic activity of UDPGT of the transplanted hepatocytes. Nocturnal phototherapy was continued throughout the studied period. Total bilirubin was considered a reliable marker of allogenic cell function. There was no significant variation of vital signs nor complications during the infusion. Mean ± SD bilirubin level was 530 ± 38 μmol/L before and 359 ± 46 μmol/L after AHT (t-test, p < 0.001). However, the introduction of phenobarbitone was followed by a drop of tacrolimus level with increase of alanine aminotransferase (ALT) and increase of bilirubin. After standard treatment of cellular rejection bilirubin fell again but from then on it was maintained at a greater level. After discharge the patient experienced a further increase of bilirubin that returned to predischarge levels after readmission to the hospital. This was interpreted as poor compliance with phototherapy. Only partial correction of clinical jaundice and the poor tolerability to nocturnal phototherapy led the parents to refuse further hepatocyte infusions and request an orthotopic liver transplant. After 24 months the child is well, with good liver function on tacrolimus and prednisolone-based immunosuppression. Isolated AHT, though effective and safe, is not sufficient to correct CN1. Maintenance of adequate immunosuppression and family compliance are the main factors hampering the success of this procedure.

Isolation of infectious Zika virus from saliva and prolonged viral RNA shedding in a traveller returning from the Dominican Republic to Italy, January 2016

We report the isolation of infectious Zika virus (ZIKV) in cell culture from the saliva of a patient who developed a febrile illness after returning from the Dominican Republic to Italy, in January 2016. The patient had prolonged shedding of viral RNA in saliva and urine, at higher load than in blood, for up to 29 days after symptom onset. Sequencing of ZIKV genome showed relatedness with strains from Latin America.

Combined HSV-TK/IL-2 gene therapy in patients with recurrent glioblastoma multiforme: biological and clinical results

Following our pilot clinical study of combined IL-2/HSV-TK gene therapy for recurrent glioblastoma multiforme (GBM), we extended the protocol to a larger population of patients and evaluated safety, feasibility, and biological activity of treatment. A total of 12 patients received intratumor injection of retroviral vector-producing cells (RVPCs), followed by intravenous ganciclovir (GCV). Treatment was well tolerated with only minor adverse events. Transduction of tumor cells was demonstrated in tumor biopsies. A marked and persistent increase of intratumor and plasma Th1 cytokine levels was demonstrated after RVPC injection. At magnetic resonance imaging evaluation, two patients had a partial response (including a patient showing disappearance of a distant noninjected tumor mass), four had a minor response, four had stable disease, and two had progressive disease. The 6- and 12-month progression-free survival rates were 47 and 14%, respectively. The 6- and 12-month overall survival rates were 58 and 25%, respectively. In conclusion, the results of our clinical protocol of gene therapy for recurrent GBM, based on combined delivery of a suicide and a cytokine gene, demonstrate that intratumor injection of RVPCs was safe, provided effective transduction of the therapeutic genes to target tumor cells, and activated a systemic cytokine cascade, with tumor responses in 50% of cases.

Infection dynamics in a traveller with persistent shedding of Zika virus RNA in semen for six months after returning from Haiti to Italy, January 2016.

We describe the dynamics of Zika virus (ZIKV) infection in a man in his early 40s who developed fever and rash after returning from Haiti to Italy, in January 2016. Follow-up laboratory testing demonstrated detectable ZIKV RNA in plasma up to day 9 after symptom onset and in urine and saliva up to days 15 and 47, respectively. Notably, persistent shedding of ZIKV RNA was demonstrated in semen, still detectable at 181 days after onset.

Excretion of West Nile Virus in Urine During Acute Infection

Detection of West Nile virus (WNV) RNA in urine has been anecdotally described and proposed for the diagnosis of WNV infection. This study reports the routine use of real-time reverse-transcription polymerase chain reaction for the detection of WNV RNA in urine to support diagnosis of WNV infection during the large outbreak that occurred in northeastern Italy in 2012. Fourteen of 32 patients (43.8%) with symptomatic WNV infection, defined as neuroinvasive disease and fever, had detectable WNV RNA in urine at the time of diagnosis, at a higher rate and load and for a longer time than detection of WNV RNA in blood. Detection of WNV RNA in urine was less frequent (2 of 14 patients [14.2%]) in blood donors in whom WNV infection was identified by WNV nucleic acid amplification testing. Infectious virus was isolated from the urine of a patient with neuroinvasive disease and a high WNV RNA load in urine.

Next-generation sequencing technologies in diagnostic virology

The data deluge produced by next-generation sequencing (NGS) technologies is an appealing feature for clinical virologists that are involved in the diagnosis of emerging viral infections, molecular epidemiology of viral pathogens, drug-resistance testing, and also like to do some basic and clinical research. Indeed, NGS platforms are being implemented in many clinical and research laboratories, as the costs of these platforms are progressively decreasing. We provide here some suggestions for virologists who are planning to implement a NGS platform in their clinical laboratory and an overview on the potential applications of these technologies in diagnostic virology.

Human papillomavirus genotyping by 454 next generation sequencing technology.

BACKGROUND An accurate tool for human papillomavirus (HPV) typing is important both for management of patients with HPV infection and for surveillance studies. OBJECTIVES Design and evaluation of an HPV typing method based on 454 next generation sequencing (NGS) technology. STUDY DESIGN Development of an HPV typing method based on 454 NGS of HPV L1 amplicons generated with MY09/11-based primers. Evaluation of the NGS method in control samples and in a panel of cervical cytological samples. Comparison of the NGS typing method with cycle sequencing and with the reverse hybridization-based INNO-LiPA HPV Genotyping Extra assay (LiPA). RESULTS In control samples carrying mixtures of HPV16 and HPV18 DNA, the NGS method could reliably detect genotype sequences occurring at a frequency of 1% in multiple infections with a sensitivity of 100 genome equivalents/μL. In cervical cytology samples, comparison with cycle sequencing demonstrated accuracy of HPV typing by NGS. The NGS method had however lower sensitivity for some HPV types than LiPA, conceivably due to the poor sensitivity of the MY09/11-based primers. At variance, LiPA could not detect HPV types which were present in low proportion in multiple infections (<10% of HPV reads obtained by NGS). In addition, NGS allowed identifying the presence of different variants of the same HPV type in a specimen. CONCLUSIONS NGS is a promising method for HPV typing because of its high sensitivity in multiple infection and its potential ability to detect a broad spectrum of HPV types, subtypes, and variants.

KPC-mediated resistance in Klebsiella pneumoniae in two hospitals in Padua, Italy, June 2009-December 2011: massive spreading of a KPC-3-encoding plasmid and involvement of non-intensive care units

BackgroundKlebsiella pneumoniae carbapenemases (KPCs) producing bacteria have emerged as a cause of multidrug-resistant nosocomial infections worldwide. KPCs are plasmid-encoded enzymes capable of hydrolysing a broad spectrum of beta-lactams, including carbapenems and monobactams, therefore worryingly limiting antimicrobial treatment options. Analysis of circulating bacterial strains and KPC alleles may help understanding the route of KPC dissemination and therefore help containing the infection.MethodsKPC-producing Klebsiella pneumoniae dissemination in two 1580- and 300- bed hospitals in Padua, Italy, from initial outbreak in 2009 to late 2011 was analysed. Molecular and clinical epidemiology, including bacterial strains, KPC-encoding plasmid sequences and associated resistance genes, involved hospital wards and relocation of patients were described. Routine antimicrobial susceptibility testing and MIC of carbapenems on clinical isolates were performed. Detection of resistance genes was obtained by PCR and sequencing. MLST, PFGE and ERIC were used for molecular genotyping. Plasmid analysis was obtained by digestion with restriction enzymes and deep sequencing.ResultsKPC-positive clinical samples were isolated from nearly 200 patients. In the initial outbreak intensive care units were almost exclusively involved, while medical, surgical and long-term wards were successively massively concerned. Analysis of KPC alleles, plasmids and bacterial sequence types (STs) indicated that during the initial outbreak KPC-3 in ST258 and KPC-2 in ST147 were each confined in one of the two surveilled hospitals. While KPC-2 dissemination was effectively contained, KPC-3 in ST258 cross-spreading was observed. The simultaneous presence of two carbapenemases, VIM-1 and KPC-2, in the same isolate was also observed in three patients. Total sequencing of plasmid content of two KPC-3 strains showed novel association of resistance plasmids.ConclusionsThe acquired molecular epidemiology demonstrated that 1) both acquisitions from outward sources and patient relocation within the hospitals were responsible for the observed spreading; 2) KPC-3-encoding Klebsiella pneumoniae ST258 prevailed over other strains. In addition, the described massive transfer of KPC-mediated resistance to non-intensive care units may anticipate spreading of resistance to the non-hospitalized population. Therefore, genotypic analysis alongside phenotypic identification of carbapenemase producers, also at the carriage state, is advisable to prevent and contain further carbapenemase resistance dissemination.

Distribution of human papillomavirus types in the anogenital tract of females and males.

Human papillomavirus (HPV) infection is the most common sexually transmitted infection in both men and women, but there are limited data comparing the prevalence of HPV infection between genders and in different anogenital sites. This cross‐sectional analysis describes the distribution of HPV types in the genital tract of 3,410 consecutive females and 1,033 males undergoing voluntary screening for HPV and referred to a single institution. The relationship between specific HPV types and the presence of anogenital lesions was examined. In both females and males, the overall prevalence of HPV infection was about 40%. A wide variety of HPV types was identified, but the prevalence of different types was remarkably similar in the two genders, even when considering different anatomical sites. HPV‐6 was the most frequent (prevalence 13%) type in all anogenital sites in men followed by HPV‐16 (7%), while HPV‐16 was the most common type in women (about 6%), either in the cervix, vagina, or vulva, followed by HPV‐6. In addition to HPV‐16, HPV‐58, HPV‐33, HPV‐31, and HPV‐56 were the carcinogenic types detected most commonly and were significantly associated with high‐grade squamous intraepithelial cervical lesions, while HPV‐53 and HPV‐66 were the most common among possibly carcinogenic types. In both genders, anogenital warts were associated with HPV‐6 and HPV‐11 infection, and, less frequently, with other types, like HPV‐54, HPV‐62, and HPV‐66. These results show that genital HPV infection involves numerous HPV types, which have similar distribution patterns in females and males and in different anogenital anatomical sites. J. Med. Virol. 82:1424–1430, 2010.

Viral invasion of the amniotic cavity (VIAC) in the midtrimester of pregnancy

Introduction: The prevalence of viral infections in the amniotic fluid (AF) has not yet been ascertained. The aim of this study was to determine the prevalence of specific viral nucleic acids in the AF and its relationship to pregnancy outcome. Study design: From a cohort of 847 consecutive women undergoing midtrimester amniocentesis, 729 cases were included in this study after exclusion of documented fetal anomalies, chromosomal abnormalities, unavailability of AF specimens and clinical outcomes. AF specimens were tested by quantitative real-time PCR for the presence of genome sequences of the following viruses: adenoviruses, herpes simplex virus (HSV), varicella zoster virus (VZV), human herpesvirus 6 (HHV6), human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), parvovirus B19 and enteroviruses. Viral nucleic acid testing was also performed in maternal blood and cord blood in the population of women in whom AF was positive for viruses and in a control group of 29 women with AF negative for viral nucleic acids. The relationship between the presence of viruses and pregnancy and neonatal outcome was examined. The correlation between the presence of nucleic acids of viruses in the AF and the concentration of the cytokine interleukin-6 (IL-6) and the T cell chemokine CXCL-10 (or IP-10) in AF and maternal blood were analyzed. Results: Viral genome sequences were found in 16 of 729 (2.2%) AF samples. HHV6 was the most commonly detected virus (7 cases, 1.0%), followed by HCMV (6 cases, 0.8%), parvovirus B19 (2 cases, 0.3%) and EBV (1 case, 0.1%), while HSV, VZV, enteroviruses and adenoviruses were not found in this cohort. Corresponding viral DNA was also detected in maternal blood of six out of seven women with HHV6-positive AF and in the umbilical cord plasma, which was available in one case. In contrast, viral DNA was not detected in maternal blood of women with AF positive for parvovirus B19, HCMV, EBV or of women with AF negative for viruses. HHV6 genome copy number in AF and maternal blood was consistent with genomic integration of viral DNA and genetic infection in all women. There was no significant difference in the AF concentration of IL-6 and IP-10 between patients with and without VIAC. However, for HCMV, there was a significant relationship between viral copy number and IP-10 concentration in maternal blood and AF. The group of women with AF positive for viral DNA delivered at term healthy neonates without complications in 14 out of 16 cases. In one case of HHV6 infection in the AF, the patient developed gestational hypertension at term, and in another case of HHV6 infection in the AF, the patient delivered at 33 weeks after preterm premature rupture of membranes (PPROM). Conclusion: Viral nucleic acids are detectable in 2.2% of AF samples obtained from asymptomatic women in the midtrimester. HHV6 was the most frequently detected virus in AF. Adenoviruses were not detected. Vertical transmission of HHV6 was demonstrated in one case.