This study reports the presence of dengue virus RNA in longitudinally collected semen samples of a previously healthy Caucasian man, returning to Italy from Thailand with primary dengue fever, up to 37 days post-symptom onset, when viraemia and viruria were undetectable. This finding, coupled with the evidence of dengue virus negative-strand RNA, an indirect marker of ongoing viral replication, in the cellular fraction of semen, indicates a need to further investigate possible sexual transmission.
Abstract Chikungunya fever is caused by Chikungunya virus (CHIKV) and is generally considered a self-limiting disease. However, severe clinical presentations with a high mortality rate have been reported in association with underlying medical conditions. This study reports the molecular characterization of the virus and an abnormal pattern of circulating cytokines in a unique lethal CHIKV case during the 2017 outbreak in Italy, which involved an elderly patient with underlying cardiac disease. Analysis of inflammatory cytokines revealed a strong increase of interferon (IFN)-α and IFN-β, as well as interleukin-6, suggesting a possible role of type-I IFN in the cytokine storm, which may be correlated with unfavorable prognosis of CHIKV infection.
To the Editor: The biologic role of amino acid variants at position 222 of the hemagglutinin (HA) gene of pandemic (H1N1) 2009 virus in severe infections has been extensively discussed. A recent series of studies (1–3) confirm the initial suggestions that G or N in this position might confer greater pathogenic potential to the virus than to the wild type. In contrast, their data suggest that no particular pathogenicity is associated with the 222E variant because it occurs at the same frequency in severe and mild infections. Most authors also seem to agree that D222G or N appears sporadically in phylogenetically distant viruses, with limited transmissibility.
However, Puzelli et al. (4) reported transmission of a 222G mutant from son to father (with the appearance of an additional G155E mutation). In Italy, the pattern of D222 variants has been peculiar, with extremely rare appearances of 222G and high diffusion of 222E isolates. At the National Institute for Infectious Diseases in Rome, 82 isolates (GenBank accession nos. {type:entrez-nucleotide-range,attrs:{text:CY063455-CY063469,start_term:CY063455,end_term:CY063469,start_term_id:296238920,end_term_id:296238948}}CY063455-CY063469 for new sequences in this study) were monitored for D222 variants. No 222G or N variants were detected, even in 24 severe infections, nor was the G155E mutation detected. This finding was not surprising, given the worldwide low frequency of this mutation, even in severe infections.
Conversely, D222E was detected in 12 of the 82 cases, peaking in September 2009, when it was present in most of the infections, with no overrepresentation in severe cases. Subsequently, it was substituted by different 222D viruses during the autumn–winter outbreak. The analysis of publicly available sequences from other centers in Italy confirmed the trend: 222E was the dominant variant during the summer, 222G was detected only in 4 cases, and 222N was never detected. As we previously reported (5), phylogenetic analysis of 222E variants allowed identification of them as an authentic circulating subclade of clade 7 (6) or cluster 2 (7), rather than as sporadically occurring variants.
To further investigate the origin and the evolution of 222 variants, we have extended the phylogenetic analysis (neighbor-joining) to 2,492 complete HA sequences from the Global Initiative on Sharing All Influenza Data database (expanded Figure online, www.cdc.gov/EID/content/17/4/749-F.htm), confirming the clustering of all the worldwide 222E variants in a well-defined subclade (Figure; expanded Figure online). The same analysis showed that D222G variants could be reconciled with 2 different phylogenetic patterns. The first less frequent pattern includes sequences appearing sparsely throughout the tree, confirming the mentioned hypothesis of sporadic mutation. In contrast, the second pattern (the majority) relates to small groups of sequences appearing in monophyletic microclusters. Among these microclusters, 2 are particularly interesting because they include only 222G sequences, isolated in different parts of the world (Figure). This finding is still compatible with sporadic mutation; bootstrap values are low because of the low general variability of these sequences. However, the possibility that D222G variants are transmissible and might sustain small epidemics of their own or that they might arise more easily from specific, phylogenetically related backgrounds, is intriguing. In a few countries, such as Italy, Norway, or Sweden, where the 222E virus has been circulating as a substantial proportion of the total virus, the 222G variants appeared more frequently in the genetic context of the 222E virus (1,4), as demonstrated by phylogenetic analysis and confirmed by the analysis of codon 239 (the codon determining the 222 residue specificity): GAA to GGA (E to G), instead of GAT to GGT (D to G). In these cases, the correct definition of 222G variants would therefore be E222G rather than D222G. From this point of view, the 222N variant would have a higher genetic barrier to change from E because it would require 2 mutations (GAA to AAT) instead of 1 (GAT to AAT), and indeed none of the 16 available (worldwide) 222N full-length variants clustered with the 222E virus. On the basis of these findings, 2 different amino acids, D and E, might be considered polymorphic variants at position 222, and the potentially more pathogenic mutants or circulating variants would be G or N.
Figure
Phylogenetic relationships among 2,492 complete hemagglutinin (HA) genes of pandemic (H1N1) 2009. At the center, the whole neighbor-joining tree. On the left, enlargement of 2 regions of the tree including pure monophyletic D222G clusters, indicated in ...
Purpose: Chikungunya fever is a mosquito-borne viral disease caused by Chikungunya virus (CHIKV) and is generally considered self-limiting non-fatal disease. However, severe clinical presentations with high mortality rate (48%) are reported associated with the presence of several underlying medical conditions. Recently, a CHIKV epidemic occurred in Italy, involving 270 confirmed cases in Lazio and Calabria regions. Here, we report the virus characterization and an abnormal pattern of circulating cytokines in a lethal case of CHIKV during the 2017 Lazio region outbreak. Methods & Materials: In September 2017, a 77-year-old male with underlying cardiac diseases was admitted for acute neurological syndrome to the Santa Maria Goretti Hospital in Latina, Rome, and he died after 9 hour admission in sub-intensive unit care for acute cardiac arrest. Due to the ongoing CHIKV outbreak, RT-PCR and indirect immunofluorescence assays were performed to assess a possible infection. The virus was isolated on BHK-21 cells, and the near-complete genome sequencing was performed. Circulating IFNs and inflammatory cytokines were evaluated and quantified using ELISA, and the levels compared to those detected during the very early stages of the infection in four non-fatal cases belonging to the same outbreak. Results: Laboratory tests showed CHIKV infection, based on positive RT-PCR test (Ct: 12), in the absence of CHIKV-specific antibodies. The sequence of the isolate (CHIKV/ITA/Lazio-INMI2-2017) showed only one nucleotide difference (synonymous substitution) as compared to the outbreak prototype strain (CHIKV/ITA/Lazio-INMI1-2017), and clustered with recent isolates of the Indian Ocean sublineage of ECSA in the maximum-likelihood phylogenetic tree. The evaluation of the inflammatory response showed that IFN-α, IFN-β and IL-6 levels were extremely high as compared to non-fatal cases, while IFN-γ and TNF-α levels were in the same range. The elevated levels of IFN-α, IFN-β and IL-6 seemed not directly correlated to elevated levels of CHIKV-RNA. Conclusion: The analysis of inflammatory cytokines revealed a remarkable and strong increase of circulating type I IFN, as well as of the IL-6 pro-inflammatory cytokine, suggesting a possible role of type I IFN in cytokine storm, which may be correlated with unfavourable prognosis of CHIKV infection.
The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unclear. We report the detection of viral RNA from different anatomical districts and the antibody profile in the first 2 COVID-19 cases diagnosed in Italy. We tested for SARS-CoV-2 RNA clinical samples, either respiratory and nonrespiratory (ie, saliva, serum, urine, vomit, rectal, ocular, cutaneous, and cervico-vaginal swabs), longitudinally collected from both patients throughout the hospitalization. Serological analysis was carried out on serial serum samples to evaluate IgM, IgA, IgG, and neutralizing antibody levels. SARS-CoV-2 RNA was detected since the early phase of illness, lasting over 2 weeks in both upper and lower respiratory tract samples. Virus isolate was obtained from acute respiratory samples, while no infectious virus was rescued from late respiratory samples with low viral RNA load, collected when serum antibodies had been developed. Several other specimens came back positive, including saliva, vomit, rectal, cutaneous, cervico-vaginal, and ocular swabs. IgM, IgA, and IgG were detected within the first week of diagnosis, with IgG appearing earlier and at higher titers. Neutralizing antibodies developed during the second week, reaching high titers 32 days after diagnosis. Our longitudinal analysis showed that SARS-CoV-2 RNA can be detected in different body samples, which may be associated with broad tropism and different spectra of clinical manifestations and modes of transmission. Profiling antibody response and neutralizing activity can assist in laboratory diagnosis and surveillance actions.
Acute hepatitis B infection (AHB) is still a common viral acute hepatitis worldwide. As vaccination, antiviral treatment, and immigration are bound to affect the epidemiological landscape of HBV infections, and some of its aspects need to be investigated: (1) the circulation of vaccine escape mutants and of primary drug resistant strains; (2) the change in HBV genotype prevalence; and (3) the clinical implications of AHB and the probability of chronification. The serological, virological, and clinical parameters of 75 patients, acutely infected by HBV, were gathered for a retrospective study. Long-term follow up, either to complete seroconversion or for up to five years, was possible for 44 patients. Sequence analysis of the reverse transcriptase/HBsAg and precore regions was performed to investigate the molecular epidemiology and pathogenesis of recent infections by HBV. Genotype distribution in AHB in Italian patients was radically different from that of chronic infections, with a dramatic increase of extra-European genotypes (A1, F), suggesting that a proportion of AHBs are currently related to imported strains. None of the documented infections occurred in vaccinated individuals, while HBsAg variants (potentially vaccine escape variants) were rare and less prevalent than in chronic infections. No drug resistant strains were observed. Spontaneous viral clearance occurred in all but three cases. Time to viral clearance was inversely proportional to liver damage, but HBsAg titer on day 28 and, better still, HBsAg decay from day 0 to day 28 after admission, were the best predictors of chronification. They are, thus, potentially useful to guide antiviral treatment to prevent chronic evolution.
ABSTRACT We recently set up a gamma interferon (IFN-γ) enzyme-linked immunospot assay (ELISPOT), using selected early secreted antigenic target 6 (ESAT-6) peptides, that appears specific for active tuberculosis (A-TB). However, ELISPOT is difficult to automate. Thus, the objective of this study was to determine if the same selected peptides may be used in a technique more suitable for routine work in clinical laboratories, such as whole-blood enzyme-linked immunosorbent assay (WBE). For this purpose, 27 patients with A-TB and 41 control patients were enrolled. Our WBE, using the already described selected peptides from ESAT-6 plus three new ones from culture filtrate protein 10, was performed, and data were compared with those obtained by ELISPOT. Using our selected peptides, IFN-γ production, evaluated by both WBE and ELISPOT, was significantly higher in patients with A-TB than in controls ( P < 0.0001). Statistical analysis showed a good correlation between the results obtained by WBE and ELISPOT ( r = 0.80, P < 0.001). To substantiate our data, we compared our WBE results with those obtained by QuantiFERON-TB Gold, a whole-blood assay based on region of difference 1 (RD1) overlapping peptides approved for TB infection diagnosis. We observed a slightly higher sensitivity with QuantiFERON-TB Gold than with our WBE (89% versus 81%); however, our test provided a better specificity result (90% versus 68%). In conclusion, results obtained by WBE based on selected RD1 peptides significantly correlate with those generated by ELISPOT. Moreover, our assay appears more specific for A-TB diagnosis than QuantiFERON-TB Gold, and thus it may represent a complementary tool for A-TB diagnosis for routine use in clinical laboratories.
