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This letter to the editor describes the case of a patient with mediastinal germ cell tumor who developed COVID-19 at the end of the first cycle of chemotherapy and continues the discussion on whether it is best to continue or delay administration of chemotherapy with an active COVID-19 infection.

Background The risk of life-threatening complications, such as visceral disseminated varicella zoster virus (VZV) infection, is greater in immunosuppressed individuals, such as systemic lupus erythematosus (SLE) patients. Case presentation Here, a case is reported of a Caucasian woman diagnosed with lupus nephritis and anti-phospholipid syndrome, who was subjected to mycophenolate mofetil and high-dose steroid remission-induction therapy. Two months later she developed abdominal pain followed by a fatal rapid multi-organ failure. As no typical skin rashes were evident, death was initially attributed to catastrophic anti-phospholipid syndrome. However, autopsy and virological examinations on archival material revealed a disseminated VZV infection. Conclusions Overall, this case highlights the importance of having a high clinical suspicion of fatal VZV infections in heavily immunosuppressed SLE patients even when typical signs and symptoms are lacking.

Aim: Plasma Gas6 was tested as an alternative to Baveno VI criteria (liver stiffness 150 × 109/l) in an endoscopy-sparing strategy. Methods: A total of 160 patients with chronic hepatitis C and advanced fibrosis/cirrhosis underwent, on the same occasion, liver elastography, upper endoscopy, a platelet count and serum Gas6 measurement. Results: A total of 74/160 (46%) patients had esophageal varices, that were small (diameter

TaqMAMA is an allele-specific PCR-based (ASPCR) method that may be suitable for broad and cost-effective genotyping applications in all types of laboratories. There is evidence that interactions between some toll like receptors (TLRs) with viruses influence both the immune response and outcome of HCMV infection. We developed a TaqMAMA genotyping assay for the detection of rs352140 TLR9 polymorphism in transplant recipients with and without HCMV infections. Performance parameters to ensure a solid pre-validation protocol have been here argued. We analysed a population of 74 kidney transplants recipients subdivided in 58 HCMV PCR positive and 16 HCMV PCR negative in the post-transplant routine control. All 74 samples were tested with 31/74 (41.9%) homozygotes (11 CC and 20 TT) and 43/74 (58.1%) heterozygotes (CT). Our preliminary data suggest that there is no correlation between TLR9 rs352140 polymorphism and frequency of HCMV infection.

Background Interferon signature (IS) is the measure of transcripts belonging to pathways of interferon activation. Viral infections can interfere with the interferon pathway, in particular herpesvirus present in immunocompromised hosts. The aim of our study was to evaluate if herpesvirus infections in immunocompromised patients with lower respiratory tract infections (LRTI) could lead to IS alterations. Methods We measured IS transcription of six genes on bronchoalveolar lavage of immunocompromised patients with LRTI (IFI27, IFI44, IFIT1, ISG15, RSAD2, SIGLEC1). Patients were divided in three groups based on Epstein-Barr virus (EBV) and other herpesviruses coinfections. Results We included 56 patients, 10 without and 17 with only EBV reactivation (respectively N and E groups) and 29 with EBV and other herpesviruses (group C). IS was higher in group C (P=0.01) compared to other ones, but single gene expressions were different among groups: IFI27 was higher whereas IFIT1 and ISG15 were lower in group C (P Conclusions The continuous stimulation of interferon cascade by herpesviruses enhances IS. The analysis of IS in immunocompromised population is possible by limiting the use of IFI27, IFIT1, ISG15 genes. Our preliminary results seem to indicate that IS is a useful biomarker of cellular response to herpesvirus infection in immunocompromised patients.

Background: The aetiology of lower respiratory tract infections is challenging to investigate. Despite the wide array of diagnostic tools, invasive techniques, such as bronchoalveolar lavage (BAL), are often required to obtain adequate specimens. PneumoniaCheckTM is a new device that collects aerosol particles from cough, allowing microbiological analyses. Up to now it has been tested only for bacteria detection, but no study has investigated its usefulness for virus identification. Methods: In this pilot study we included 12 consecutive patients with pneumonia. After testing cough adequacy via a peak flow meter, a sampling with PneumoniaCheckTM was collected and a BAL was performed in each patient. Microbiological analyses for virus identification were performed on each sample and concordance between the two techniques was tested (sensitivity, specificity and positive/negative predictive values), taking BAL results as reference. Results: BAL was considered adequate in 10 patients. Among them, a viral pathogen was identified by PneumoniaCheckTM 6 times, each on different samples, whereas BAL allowed to detect the presence of a virus on 7 patients (14 positivities). Overall, the specificity for PneumoniaCheckTM to detect a virus was 100%, whereas the sensitivity was 66%. When considering only herpes viruses, PneumoniaCheckTM showed a lower sensitivity, detecting a virus in 1/4 of infected patients (25%). Conclusions: In this pilot study PneumoniaCheckTM showed a good correlation with BAL for non-herpes virologic identification in pneumonia patients, providing excellent specificity. Further studies on larger population are needed to confirm these results and define its place in the panorama of rapid diagnostic tests for lower respiratory tract infections.

It is known that several viruses can alter or modulate the transcriptional and translational activity of host cells to obtain a rapid and efficient replication. In particular, Human Cytomegalovirus (HCMV) can interact with host cell at multiple levels, even modulating the expression of small signaling molecules called microRNAs. Especially, human miRNA mir-146a expression seems to be downregulated by HCMV infection in vitro. The aim of this study was to evaluate mir-146a expression in kidney transplant patients during HCMV infection. Sixty-four serum samples from 22 kidney transplant patients were analyzed and subdivided in three groups (high viral load, low viral load, and absent viral load). Mir-146a expression for each sample has been evaluated by a specific stem-loop Real Time polymerase chain reaction, and a statistical analysis was performed. Expression levels of mir-146a were similar among the three groups tested showing no statistical significant difference. Results obtained did not confirm data previously reported in literature, but the change of mir-146a expression levels has to be more clearly defined as it could not be directly caused by virus replication.

According to the latest update, 2,578 unique mature micro­RNAs (miRNAs) are currently annotated in the human genome and participate in the regulation of multiple events, such as cellular proliferation or apoptosis. A previous study analyzing global miRNA expression patterns in GH cells (high human endogenous retrovirus, HERV, K vs. low) showed that 2 miRNAs (miR-663 and miR-638) are differentially regulated and exhibit expression parallel to that of HERV-K. The aim of this study was to evaluate HERV-K and -W pol gene and miR-155 expression in kidney transplant recipients and the possible relationship between them. The comparison between kidney transplant patients negative for human cytomegalovirus (HCMV) infection and positive patients showed a significant difference in terms of miR-155 expression (p = 0.0111). We demonstrated that HERV-K and -W pol gene expression was significantly higher in CMV-infected kidney transplant recipients versus those not infected as previously reported by our groups. Our correlation data suggest that miR-155 are not directly involved in regulating the HERV notwithstanding that we together observed increased expression of HERV-K and -W and diminished expression of miR-155 in HCMV-infected human kidney transplant recipients.

Background Human endogenous retrovirus (HERVs) constitute approximately 8% of the human genome. Induction of HERV transcription is possible under certain circumstances, and may have a possible role in some pathological conditions. Objectives The aim of this study was to evaluate HERV-K and –W pol gene expression in kidney transplant recipients and to investigate the possible relationship between HERVs gene expression and CMV infection in these patients. Study design Thirty-three samples of kidney transplant patients and twenty healthy blood donors were used to analyze, HERV-K and –W pol gene RNA expression by relative quantitative relative Real-Time PCR. Result We demonstrated that HERVs pol gene expression levels were higher in kidney transplant recipients than in healthy subjects. Moreover, HERV-K and –W pol gene expression was significantly higher in the group of kidney transplant recipients with high CMV viral load than in the groups with no or moderate CMV viral load. Conclusion Our data suggest that CMV may facilitate in vivo HERV activation.

MicroRNAs are small, noncoding RNAs that control expression of target genes through inhibiting protein translation or inducing degradation of mRNA transcripts of target genes. According to the latest update, 2578 unique mature miRNAs are currently annotated in the human genome and participate in the regulation of multiple events, such as cellular proliferation or apoptosis. Herpesvirus family comprehends many viruses able to control and modulate host-cell processes permitting the survival by a latency phase after primary infection. Recently has been attested that Human Cytomegalovirus, which belongs to Herpesvirus family, can alter human miRNAs expression in vitro, and, in particular, downregulate mir-155 expression. In this study 20 kidney transplant patients positive to Human Cytomegalovirus infection and 11 negative were enrolled. The patients' positive to Human Cytomegalovirus infections have been subdivided into two groups: one group including patients with a low viral load and one including patients with a high viral load. The mir-155 expression profile has been evaluated by a stem-loop Real Time PCR in all these conditions to observe differences among the groups and compare the results obtained with the literature. The comparison between kidney transplant patients negative to Human Cytomegalovirus infection and patients with a high viral load showed a not significant difference in terms of mir-155 expression. However, considering low viral load group or the group including both high and low viral load patients, mir-155 expression levels decreased significantly. Considering this data together, it is possible confirm data published before and assert that Human Cytomegalovirus is responsible of mir-155 downregulation.

Per POCT (point of care testing) s’intende l’analisi eseguita vicino al punto di cura del paziente, con il presupposto che il risultato sia disponibile immediatamente o in un lasso di tempo molto breve, al fine di permettere ai clinici una diagnosi rapida e/o una rapida decisione terapeutica. Fino a poco tempo fa tra gli esami effettuati in un POCT non figuravano test di biologia molecolare per la diagnosi delle infezioni virali, a causa della complessita dei sistemi analitici e delle procedure esecutive di questi test. Negli ultimi anni, pero, e risultata sempre piu evidente la necessita di diagnosi rapide e semplici anche in questo settore, al fine di permettere decisioni terapeutiche immediate. Una di queste necessita riguarda la diagnosi delle meningiti e meningoencefaliti virali causate da enterovirus (EV). Un corretto e tempestivo riconoscimento della causa eziologica permetterebbe una terapia piu adeguata per i pazienti. Lo scopo di questo studio era confrontare i dati ottenuti dall’utilizzo di una strumentazione POCT per la diagnosi molecolare di infezione da enterovirus con i dati derivati dal procedimento operativo attualmente utilizzato dal Laboratorio, al fine di comparare costi, rapidita di esecuzione e di diagnosi, semplificazione dei processi operativi, gestione del personale, possibile outcome clinico. Attualmente il Laboratorio utilizza un test in Real Time PCR qualitativo per la ricerca degli enterovirus (Enterovirus R-gene, Argene, North Massapequa, NY, USA). Lo studio ha previsto una prima fase di raccolta dei dati sui processi attualmente utilizzati dal Laboratorio (tempi e costi di esecuzione dei test, impegno del personale, difficolta operative, numero di test utili clinicamente). Successivamente questi dati sono stati confrontati con quelli di un altro test molecolare che utilizza anch’esso un metodo di Real Time PCR qualitativa (Xpert EV, Cepheid, Sunnyvale, CA, USA) con una nuova strumentazione POCT (GeneXpert, Cepheid, Sunnyvale, CA, USA), come descritti da diverse altre pubblicazioni. Il confronto tra i due metodi ha dimostrato una sensibilita e una specificita sovrapponibili. Dai risultati di questo studio, risulta che il metodo utilizzato attualmente nel nostro Laboratorio non puo essere considerato adeguato per una diagnostica d’urgenza. Al contrario, l’utilizzo del sistema GeneXpert puo portare a una significativa riduzione del tempo di risposta (da 5/6 giorni a 3 ore), del tempo di esecuzione del test (da 5 a 2,45 ore) e del tempo di esecuzione tecnica (da 2 ore a soli 10 minuti). La riduzione del tempo di ospedalizzazione (da 6,6 a 1,2 giorni per i pazienti pediatrici e da 7,3 a 1,9 giorni per gli adulti) e dell’impiego di terapie antibiotiche e antivirali non necessarie fanno di questo test un utile strumento per migliorare la gestione complessiva dei pazienti. Un ulteriore vantaggio e rappresentato dalla completa automazione del test e dalla notevole semplicita di utilizzo. Queste caratteristiche rendono questo sistema adatto a una realta di point of care utilizzabile anche da personale non specificatamente addestrato. L’analisi dei costi ha dimostrato un notevole risparmio, grazie alla diminuzione delle spese di degenza (fino a un risparmio del 33%), oltre a una riduzione sostanziale del costo del test (da €92,25 a €59,95 per test). In conclusione, i dati ottenuti indicano che questo nuovo test per enterovirus possiede le caratteristiche ottimali per una sua utile introduzione nella routine diagnostica delle urgenze virali.

In 2009, 2589 mosquitoes were collected in northwest Italy and screened for orthobunyavirus RNA by RT-PCR. One pool of Anopheles maculipennis complex mosquitoes was found to be positive and a virus was isolated from that pool. The isolate was identified as Batai virus (BATV) by sequencing. Previously, BATV was detected in Italy, but limited data and no prior isolates existed. Full-length sequences of the S, M and L segments were determined for the newly isolated Italian strain. For comparison, partial sequences were also determined for the BATV strain Calovo (former Czechoslovakia, 1960). Phylogenetic analyses revealed clustering of the newly derived Italian BATV along with a recent isolate from Germany and the historic strain Calovo. To the best of our knowledge, this represents the first isolation of BATV from Italy, which confirms a broader geographical distribution of BATV in Europe than was previously verified by isolation.

To the Editor: Dengue is a mosquito-transmitted viral disease endemic to the tropics and subtropics worldwide. It is caused by 4 dengue virus serotypes (DENV-1–4) that belong to the genus Flavivirus. The disease varies from dengue fever to life-threatening hemorrhagic fever and shock that are associated with secondary infections. During recent decades, dengue incidence and prevalence have increased in disease-endemic areas, and the disease has been increasingly recognized in travelers (1). We report a case of dengue in a man who had traveled to Yemen. In June 2010, a 38-year-old Italian man was admitted to the hospital for high fever (maximum 39.5°C) after a 1-week work-related stay in Yemen, near Mukalla, in the province of Hadhramaut. The patient had visited the countryside where he was heavily bitten by mosquitoes. On the third day after onset of fever, the patient started to experience strong and unremitting frontal and retro-orbital headache and joint pains, which lasted for 5 days. He also experienced vomiting. Laboratory test results showed mild leukopenia (2.41 × 103 cells/mm3) and lowered platelet counts (96 × 103 cells/mm3), increased liver alanine aminotransferase levels (151 U/L), and mildly abnormal blood clotting (prothrombin time, international normalized ratio 1.24). In 1 week, the patient started to recover and was discharged from the hospital. The patient received antimicrobial (levoxacin) and antipyretic (acetaminophen) drugs. Laboratory testing after discharge showed increased levels of hepatic enzymes, which reached maximum levels on day 13 after onset of symptoms (alanine aminotransferase 669 U/L) and decreased to within reference limits in 1 month. A plasma sample taken on day 6 after disease onset was positive for flavivirus RNA by reverse transcription–PCR (RT-PCR) specific for members of the genus Flavivirus (2). The RT-PCR product was sequenced, and according to BLAST (www.ncbi.nlm.nih.gov/blast), the 184-bp sequence obtained shared 99% nt identity with dengue serotype 3 viruses in GenBank. The plasma sample also had positive results for dengue virus nonstructural protein 1 (NS1) antigen test (Platelia NS1 Ag ELISA; Bio-Rad, Marnes-la-Coquette, France), anti-dengue immunoglobulin (Ig) M ELISA (Focus Technologies, Cypress, CA, USA), and in an in-house IgG immunofluorescence assay that used DENV-3–infected Vero E6 cells as antigens (titer 40). Other concomitant infections were ruled out by bacterial cultures and by laboratory tests for various viral, bacterial, and parasitic pathogens. Virus isolation was conducted as described (3). Viral RNA was extracted from the supernatant of the infected Vero E6 cells, and the envelope gene was amplified in an RT-PCR. The amplified product was directly sequenced (details available from P.R. upon request). The obtained envelope gene sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"HQ336219","term_id":"317016905","term_text":"HQ336219"}}HQ336219) of 1,479 bp was aligned with 26 other DENV-3 strains, including the most similar sequences identified in nucleotide BLAST search and a global set of sequences representing different genotypes (4), by using MUSCLE (www.ebi.ac.uk/Tools/muscle/index.html). A neighbor-joining phylogenetic tree was inferred by using p-distance, with 1,000 bootstrap replicates in MEGA version 4 (www.megasoftware.net). The strain isolated from Yemen in 2010 (this study) shared highest nucleotide homologies with strains from Jeddah, Saudi Arabia (98%–99%), and Tanzania (98%) and was phylogenetically grouped within genotype III of DENV-3. The most closely related strains also included recent isolates from Cote d’Ivoire, People’s Republic of China, Bhutan, and India (Figure). Figure Neighbor-joining phylogenetic tree based on complete envelope gene sequences of dengue virus (DENV) serotype 3 virus, rooted with DENV-1. Bootstrap support values >80 are shown. Boldface indicates the 2010 isolate from Yemen. Scale bar represents ... Dengue has been documented in the Middle East, including Saudi Arabia (5) and Yemen (6). In May 2010, a dengue outbreak in Yemen was reported (7). The patient reported here had visited Yemen in June, and by August the outbreak had resulted in ≈100,000 infections and 200 deaths (7). To our knowledge, the DENV strains involved in this outbreak had not been previously characterized. The same genotype as the isolate described here, genotype III of DENV-3, was most recently isolated from Saudi Arabia in 2005 and has been associated with recent outbreaks in Sri Lanka, East Africa, and Latin America (8). Without further information, it remains unknown whether other serotypes or genotypes circulate concurrently in Yemen. According to the Italian Public Health Institute (Istituto Superiore di Sanita), the numbers of imported dengue cases in Italy are increasing (www.iss.it/binary/publ/cont/09_11web.pdf), but because only a few hospitals perform diagnostic tests, dengue is likely to be underdiagnosed in Italy. Viremic travelers can contribute to spread of DENV, and during the active mosquito season, travelers from dengue-endemic areas who have diagnosed or suspected dengue should be advised to avoid contact with mosquitoes. Recently, indigenous transmission of dengue virus was shown to have occurred in Cote d’Azur in southern France (9) and in Croatia (10), thereby highlighting the risk in areas that have Aedes albopictus mosquitoes, which are competent DENV vectors. In these areas, including Italy, vector control and surveillance of DENV in mosquitoes should be conducted. We conclude that recognition and diagnosis of dengue in travelers should be emphasized and that characterization of DENV strains from travelers helps elucidate the molecular epidemiology of DENV in a global context.

Background Correlating human papillomavirus (HPV) type with the clinical and histopathological features of skin lesions (from genital and nongenital sites) can present a diagnostic challenge. Objective In this study, HPV infection patterns were correlated with pathology and clinical presentation in lesional and nonlesional body sites from a young patient with a primary T-cell immunodeficiency. Methods HPV infection was evaluated at both DNA and protein levels by polymerase chain reaction and immunohistochemistry. Results The patient's genital lesions were caused exclusively by α-genotypes (high-risk type HPV-51 in the anal and low-risk type HPV-72 in the penile condylomas). The opposite was true for the skin lesions, which were infected by β-genotypes alone (HPV-8 and HPV-24). HPV-24 was the predominant type in terms of viral load, and the only one found in productive areas of infection. The patient had already developed high-grade dysplasia in the anal condyloma-like lesions, and showed areas of early-stage dysplasia in the lesions caused by the β-genotype HPV-24. Limitations The basic origin of the immunodeficiency is not yet defined. Conclusion These findings provide proof of principle that both α- and β-genotypes can cause overt dysplastic lesions when immunosurveillance is lost, which is not restricted to epidermodysplasia verruciformis.

Mosquitoes collected in northern Italy were screened for flavivirus RNA. Positive amplicons were sequenced and found most similar to insect flavivirus (ISF), Usutu virus (USUV) and surprisingly also to Japanese encephalitis virus (JEV). The sequence (167 bp), obtained from one pool of Culex pipiens, was found identical to JEV strains from bats in China. Unfortunately additional sequence data or virus isolations were not obtained in this study. Confirmation of potential introduction of JEV to Italy and other European countries is urgently needed.

Alkhurma hemorrhagic fever virus (AHFV) is a tick-borne flavivirus with high case fatality rates, endemic in the Arabian Peninsula. Recently AHFV was detected in travelers returning from Egypt suggesting geographical spreading. We also report AHFV infection in a traveler ex Egypt, representing atypical symptoms of rhabdomyolysis and severe muscular weakness.

Batai virus (BATV) was identified in mosquitoes in the Caltignaga region of Novarra, northern Italy in 2009. Here, we report the identification of antibodies to BATV in serum samples that were taken from healthy bovines in that region in 2011. BATV has been associated with a mild febrile human illness and identified as the likely parental segment donor in a reassortment event that resulted in the generation of the virulent progeny, Ngari virus. The possible veterinary disease associations of BATV are unknown. The presence of antibodies to BATV in bovine populations confirms local transmission in northern Italy. Given its likely role as a segment donor, an understanding of the geographic and host distributions of BATV is of veterinary and human public health interest.

Nucleic acid detection by amplification (NAT) assays began to be used in diagnostic microbiology about twenty years ago. Since then, the progress of molecular methods has been continuous and rapid, aimed to obtain more and more sensitive and specific results and automation.To actually achieve such objectives, microbiology laboratories have to use suited quality controls and to participate to external quality control programs. Quality controls for NAT assays need to be adjusted to follow the evolution of the molecular methods.The need of standardization and suited controls for NAT assays became evident approximately by the middle of ’90 years.Thus International Standards (IS) were established by the World Health Organization, for the main blood borne viruses. Such preparations had a well known concentration expressed as International Units, a new measure unit for nucleic acid quantitation. Then, IS were used in order to calibrate working reagents and synthetic calibrators. Another tool for standardization are multicentre studies aimed to verify the performances of participating laboratories using different molecular methods, and allow the comparison of inter-laboratory results.The CoSBio of AMCLI, as other European organizations, in the last years promoted, some multicentre studies on NAT assays. The results of such studies demonstrated a good level of diagnostic performances of participating laboratories. However, with the introduction of the real time PCR (RQ-PCR) a new problem emerged: the variability of results obtained by molecular methods due to different coefficient of linear amplification. Solutions suggested by Literature to this and similar problems are examined, mainly focusing on the assessment of the Calibration curves in the RQ-PCR. Finally, the Authors signal some important fields in the molecular diagnostics, such as the detection of HPV and the detection of BKV, still lacking of sufficient standardization.

NAT (Nucleic Acid Amplification Technology) assays are now widely used in diagnostic microbiology. The implementation of NAT assays requires the use and the availability of control reagents, with various aims, such as to verify the efficiency of the procedures and the comparability of the results obtained among different laboratories. At this purpose, the Committee for the Study of Biotechnologies (CoSBio) of AMCLI performed a study to evaluate a group of synthetic quality controls for the detection of HBV-DNA, HCV-RNA, HIV-DNA e CMV-DNA, commercially distributed by AlfaWassermann. Here we report the results of the first part of the study performed to assess the main characteristics of these reagents and their applicability to different diagnostic systems. In the second part, the same controls will be evaluated in a national multicentre study.

The syndrome defined as "idiopathic CD4 lymphocytopenia' (ICL) is a rare disease of unknown aetiology, often associated with severe depression of immune defences and the occurrence of opportunistic infections. A case is reported wherein a severe immunodeficiency syndrome with persistent idiopathic CD4+ lymphopenia developed in a woman suffering from systemic microscopic polyarteritis; no signs of HIV 1/2 or HTLV I/II infection were evident. The patient died of widespread opportunistic infections. The association of ICL with vasculitis has never been reported until now. A link between the two diseases cannot be ruled out.