Rapid diagnosis of bloodstream infections in the critically ill: Evaluation of the broad-range PCR/ESI-MS technology.

Bloodstream infection (BSI) and associated sepsis represent a major source of mortality in industrialized countries. Prompt treatment with targeted antibiotics affects both the financial impact and the clinical outcome of BSI: every hour gained in initiating the correct antimicrobial therapy significantly increases the probability of patient survival. However, the current standard-of-care, which depends on blood culture-based diagnosis, are often unable to provide such a fast response. Fast and sensitive molecular techniques for the detection of sepsis-related pathogens from primary blood samples are strongly needed. The aim of this study was to assess the usefulness of the IRIDICA BAC BSI Assay, a PCR/ESI-MS-based technology for the early diagnosis of bloodstream infections from primary blood samples in critical patients. This evaluation has been performed by comparison with the traditional culture-based methods. The study was performed on a total of 300 prospective whole blood specimens obtained from patients suspected of sepsis, admitted to enrolling ER units from The Greater Romagna Area. The overall concordance between the two techniques was of 86%, with a calculated sensitivity of 76% and an assay specificity of 90%. The clinical significance of discrepant results was evaluated reviewing the patients' clinical records and the results of additional relevant microbiological tests. The data here obtained support the ability of the IRIDICA BAC BSI Assay to identify a broad range of bacteria directly from primary whole blood samples, within eight hours. This might allow a timely administration of a suitable treatment.

Serological and molecular tools to diagnose visceral leishmaniasis: 2-years' experience of a single center in Northern Italy.

The diagnosis of visceral leishmaniasis (VL) remains challenging, due to the limited sensitivity of microscopy, the poor performance of serological methods in immunocompromised patients and the lack of standardization of molecular tests. The aim of this study was to implement a combined diagnostic workflow by integrating serological and molecular tests with standardized clinical criteria. Between July 2013 and June 2015, the proposed workflow was applied to specimens obtained from 94 in-patients with clinical suspicion of VL in the Emilia-Romagna region, Northern Italy. Serological tests and molecular techniques were employed. Twenty-one adult patients (22%) had a confirmed diagnosis of VL by clinical criteria, serology and/or real-time polymerase chain reaction; 4 of these patients were HIV-positive. Molecular tests exhibited higher sensitivity than serological tests for the diagnosis of VL. In our experience, the rK39 immunochromatographic test was insufficiently sensitive for use as a screening test for the diagnosis of VL caused by L. infantum in Italy. However, as molecular tests are yet not standardized, further studies are required to identify an optimal screening test for Mediterranean VL.

Clinical application of a molecular method based on real time RT-PCR for detection of influenza A(H1N1)v virus.

Given the new diagnostic need following the pandemic caused by the A(H1N1)v virus, we evaluated the performance characteristics of Xpert® Flu assay (Cepheid). The overall sensitivity and specificity were 65.6% and 92.8%, respectively. Sensitivity and specificity for A(H1N1)v virus were 85.7% and 94.9%, respectively, and therefore the Xpert® Flu assay is suitable for a rapid diagnosis in critically ill patients where diagnosis is crucial for clinical management and for an appropriate public health response.

Diagnosis of west nile virus human infections: overview and proposal of diagnostic protocols considering the results of external quality assessment studies.

West Nile virus, genus Flavivirus, is transmitted between birds and occasionally other animals by ornithophilic mosquitoes. This virus also infects humans causing asymptomatic infections in about 85% of cases and <1% of clinical cases progress to severe neuroinvasive disease. The virus also presents a threat since most infections remain unapparent. However, the virus contained in blood and organs from asymptomatically infected donors can be transmitted to recipients of these infectious tissues. This paper reviews the presently available methods to achieve the laboratory diagnosis of West Nile virus infections in humans, discussing the most prominent advantages and disadvantages of each in light of the results obtained during four different External Quality Assessment studies carried out by the European Network for 'Imported' Viral Diseases (ENIVD).

Persistence of anti-West Nile virus-specific antibodies among asymptomatic blood donors in northeastern Italy.

The development and persistence of anti-West Nile Virus (WNV) immunoglobulin G (IgG)- and IgM-specific antibodies were investigated in 68 asymptomatic blood donors (BDs) previously tested as positive between October, 2008, and September, 2009, and living in northeastern Italy. Our study showed that WNV-specific IgG titers became negative (41%) or decreased (33%) in a large percentage of BDs, while they increased in a smaller percentage (10%); 16% of BDs showed no titer variation. Reversion to seronegative status within a short time frame suggests that WNV surveillance should be maintained year after year.

West nile virus infection in the Mesopotamia region, Syria border of Turkey.

We described the serological prevalence of West Nile Virus (WNV) antibodies among the human population in a historical and strategic region of Turkey. A serologic survey was conducted based on suspected cases in April, 2009, in the Mesopotamia region of Turkey, in the villages that were located alongside the Zergan River. All the sera were tested by enzyme-linked immunosorbent assay ELISA (Euroimmune™), and the positive samples were tested by immunofluorescent assay (IFA; Euroimmune™). As confirmation, neutralizing antibodies against WNV were tested by microneutralization assay (MNTA). In total, 307 individuals were included. The MNTA test was found to be positive among 52 individuals out of 307 (17%). In multivariate analysis, age >50 [odds ratio (OR)=5.2, confidence interval (CI) 2.76-9.97, p<0.001) and being in an occupational risk group (OR=2.02, CI 1.02-4.04, p=0.044) were found to be the risk factors for WNV seropositivity with the MNTA test. The physicians in the region should be aware of the risk of WNV infection and should be alerted to detect the clinical cases.

Comparative genomic and phylogenetic analysis of the first Usutu virus isolate from a human patient presenting with neurological symptoms.

Usutu virus (USUV) is a mosquito-borne flavivirus, belonging to the Japanese encephalitis antigenic complex, that circulates among mosquitoes and birds. We describe and analyze the complete genome sequence of the first USUV strain isolated from an immunocompromised patient with neuroinvasive disease. This USUV isolate showed an overall nucleotide identity of 99% and 96%, respectively, with the genomes of isolates from Europe and Africa. Comparison of the human USUV complete polyprotein sequence with bird-derived strains, showed two unique amino acid substitutions. In particular, one substitution (S595G) was situated in the DIII domain of the viral Envelope protein that is recognized by flavivirus neutralizing antibodies. An additional amino acid substitution (D3425E) was identified in the RNA-dependent RNA polymerase (RdRp) domain of the NS5 protein. This substitution is remarkable since E3425 is highly conserved among the other USUV isolates that were not associated with human infection. However, a similar substitution was observed in Japanese encephalitis and in West Nile viruses isolated from humans. Phylogenetic analysis of the human USUV strain revealed a close relationship with an Italian strain isolated in 2009. Analysis of synonymous nucleotide substitutions (SNSs) among the different USUV genomes showed a specific evolutionary divergence among different countries. In addition, 15 SNSs were identified as unique in the human isolate. We also identified four specific nucleotide substitutions in the 5' and 3' untranslated regions (UTRs) in the human isolate that were not present in the other USUV sequences. Our analyses provide the basis for further experimental studies aimed at defining the effective role of these mutations in the USUV genome, their potential role in the development of viral variants pathogenic for humans and their evolution and dispersal out of Africa.

Single-reaction, multiplex, real-time rt-PCR for the detection, quantitation, and serotyping of dengue viruses.

BACKGROUND: Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction. METHODOLOGY/PRINCIPAL FINDINGS: An rRT-PCR assay targeting the 5' untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log10 cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1-4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested. CONCLUSIONS/SIGNIFICANCE: This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.

Seroprevalence of West Nile virus antibodies in blood donors living in the metropolitan area of Milan, Italy, 2009-2011.

A seroprevalence study for anti-West Nile virus-specific antibodies was carried out in healthy blood donors resident in the metropolitan area of Milan in two different years, 2009 and 2011. In 2009 no positive sera were found, whereas 5 positive sera were found in 2011, revealing viral circulation in this naive area. The seroprevalence rate identified in 2011 was 0.57%, suggesting that the area of WNV circulation in Italy is larger than that previously identified.

Heterogeneity of West Nile virus genotype 1a in Italy, 2011.

West Nile virus (WNV) is currently circulating in several European countries and, over recent decades, concomitantly with enhanced surveillance studies and improved diagnostic capabilities, an increase in the geographical distribution and in the number of cases in Europe has been documented. In Italy in 2011, 14 human cases of WNV neuroinvasive infections due to lineage 1 strains were registered in several Italian regions. Here we report WNV partial sequences obtained from serum samples of two patients living in different regions of Italy (Veneto and Sardinia). Phylogenetic analysis, performed on a fragment (566 nt) of the envelope gene, showed that WNV strains circulating in Italy in 2011 belong to lineage 1a, but are different from lineage 1a strains isolated in 2008-2009.The data reported here are consistent with the hypothesis of multiple recent introductions of WNV lineage 1a strains into Italy.

Detection of West Nile virus RNA (lineages 1 and 2) in an external quality assessment programme for laboratories screening blood and blood components for West Nile virus by nucleic acid amplification testing.

BACKGROUND: A second Italian external quality assessment programme was run in 2011 to assess the performance of blood transfusion centres in detecting West Nile virus RNA in plasma. MATERIALS AND METHODS: Each participant received two panels containing negative samples and samples positive for West Nile virus lineages 1 and 2, some of which with a viral concentration close to or below the 95% limit of detection of the respective commercial nucleic acid amplification test assay: the PROCLEIX WNV assay or the Cobas TaqScreen West Nile virus test. RESULTS: Eleven laboratories took part in the external quality assessment programme. All of them correctly identified the positive samples with a viral concentration above the 95% limit of detection. No false positive results or pre-/post-analytical errors were observed. DISCUSSION: The External quality assessment programme run in 2011 allowed participants to assess the performance of the nucleic acid amplification test methods applied in their seasonal routine screening of blood donations. The results confirm the 95% limit of detection reported by the test kits' manufacturers for both West Nile virus lineages.

Mosquito, bird and human surveillance of West Nile and Usutu viruses in Emilia-Romagna Region (Italy) in 2010.

BACKGROUND: In 2008, after the first West Nile virus (WNV) detection in the Emilia-Romagna region, a surveillance system, including mosquito- and bird-based surveillance, was established to evaluate the virus presence. Surveillance was improved in following years by extending the monitoring to larger areas and increasing the numbers of mosquitoes and birds tested. METHODOLOGY/PRINCIPAL FINDINGS: A network of mosquito traps, evenly distributed and regularly activated, was set up within the surveyed area. A total of 438,558 mosquitoes, grouped in 3,111 pools and 1,276 birds (1,130 actively sampled and 146 from passive surveillance), were tested by biomolecular analysis. The survey detected WNV in 3 Culex pipiens pools while Usutu virus (USUV) was found in 89 Cx. pipiens pools and in 2 Aedes albopictus pools. Two birds were WNV-positive and 12 were USUV-positive. Furthermore, 30 human cases of acute meningoencephalitis, possibly caused by WNV or USUV, were evaluated for both viruses and 1,053 blood bags were tested for WNV, without any positive result. CONCLUSIONS/SIGNIFICANCE: Despite not finding symptomatic human WNV infections during 2010, the persistence of the virus, probably due to overwintering, was confirmed through viral circulation in mosquitoes and birds, as well as for USUV. In 2010, circulation of the two viruses was lower and more delayed than in 2009, but this decrease was not explained by the relative abundance of Cx. pipiens mosquito, which was greater in 2010. The USUV detection in mosquito species confirms the role of Cx. pipiens as the main vector and the possible involvement of Ae. albopictus in the virus cycle. The effects of meteorological conditions on the presence of USUV-positive mosquito pools were considered finding an association with drought conditions and a wide temperature range. The output produced by the surveillance system demonstrated its usefulness and reliability in terms of planning public health policies.

Toscana virus infections in northern Italy: laboratory and clinical evaluation.

Toscana virus (TOSv) is a neurotropic arthropod-borne virus that causes meningitis in the Mediterranean basin during the summer months. A total of 120 patients suffering from acute aseptic meningitis between July 1 and October 31, 2010 in northern Italy were evaluated. Eighteen of them (15%) were in the acute stage of TOSv disease.

Detection of Usutu-virus-specific IgG in blood donors from northern Italy.

We developed a novel enzyme-linked immunosorbent assay to detect the specific IgG response to Usutu virus (USUV) in humans, by evaluating 359 blood donors who were living in northeastern Italy. Our results demonstrate the presence of an anti-USUV response in 4 subjects with no history of other flavivirus infection.

Seroprevalence of West Nile virus-specific antibodies in a cohort of blood donors in northeastern Italy.

IgG and IgM levels against West Nile virus (WNV) were measured in 20,033 serum samples that were obtained between October 2008 to September 2009 from 9913 blood donors in the district of Ferrara, northeastern Italy. As confirmatory test, a microneutralization assay was used to detect the presence of neutralizing antibodies against WNV. Sixty-eight subjects (0.69%) were positive for anti-WNV by immunofluorescence assay. Large differences in the prevalence of antibodies to WNV were noted between towns in the area evaluated.

Inflammatory cytokine expression is associated with chikungunya virus resolution and symptom severity.

The Chikungunya virus infection zones have now quickly spread from Africa to parts of Asia, North America and Europe. Originally thought to trigger a disease of only mild symptoms, recently Chikungunya virus caused large-scale fatalities and widespread economic loss that was linked to recent virus genetic mutation and evolution. Due to the paucity of information on Chikungunya immunological progression, we investigated the serum levels of 13 cytokines/chemokines during the acute phase of Chikungunya disease and 6- and 12-month post-infection follow-up from patients of the Italian outbreak. We found that CXCL9/MIG, CCL2/MCP-1, IL-6 and CXCL10/IP-10 were significantly raised in the acute phase compared to follow-up samples. Furthermore, IL-1ß, TNF-α, Il-12, IL-10, IFN-γ and IL-5 had low initial acute phase levels that significantly increased at later time points. Analysis of symptom severity showed association with CXCL9/MIG, CXCL10/IP-10 and IgG levels. These data give insight into Chikungunya disease establishment and subsequent convalescence, which is imperative to the treatment and containment of this quickly evolving and frequently re-emerging disease.

Phylogenetic analysis of West Nile virus isolates, Italy, 2008-2009.

To determine the lineage of West Nile virus that caused outbreaks in Italy in 2008 and 2009, several West Nile virus strains were isolated from human specimens and sequenced. On the basis of phylogenetic analyses, the strains isolated constitute a distinct group within the western Mediterranean cluster.

A rapid and specific real-time RT-PCR assay to identify Usutu virus in human plasma, serum, and cerebrospinal fluid.

BACKGROUND: Usutu virus (USUV), a flavivirus that belongs to the Japanese encephalitis virus (JEV) family, has recently emerged as a human pathogen, necessitating new diagnostic tools. OBJECTIVE: The development and assessment of a real-time RT-PCR assay to detect USUV in human samples. STUDY DESIGN: Based on USUV genomic sequences from GenBank, USUV-specific primers and probes that target the NS5 gene were designed. The sensitivity was evaluated in a 10-fold dilution series of plasmid that contained the amplicon and in a dilution series of a quantified human USUV isolate. The specificity was determined by testing various concentrations of related ArBo viruses, including flaviviruses and phleboviruses. Human RNAse P was also amplified in the assay. One hundred four human specimens from patients who suffered from viral meningoencephalitis were evaluated. RESULTS: The real-time RT-PCR assay had a sensitivity of 50 genomic copies per reaction (corresponding to 2200 copies/ml) and 1 PFU/ml of USUV isolate. USUV isolates from Austria were identified with identical efficiency, and no ArBo viruses, other than USUV, were detected. USUV was also identified in 3 cerebrospinal fluid samples. All human samples were positive for RNAse P. CONCLUSIONS: This PCR assay is recommended for all cases in which a rapid and clinically accurate diagnosis of human USUV infection is required.

False-positive transcription-mediated amplification assay detection of West Nile virus in blood from a patient with viremia caused by an Usutu virus infection.

Detection of West Nile virus (WNV) by nucleic acid amplification technology (NAAT) is used widely to screen blood and organ donations in areas where WNV is endemic. We report a false-positive result of a WNV transcription-mediated amplification assay (TMA) in a patient with viremia that was caused by Usutu virus, a mosquito-borne flavivirus.

Absence of neuroinvasive disease in a liver transplant recipient who acquired West Nile virus (WNV) infection from the organ donor and who received WNV antibodies prophylactically.

We describe the first case of West Nile virus (WNV) infection in Europe with transmission from donor to recipient following liver transplantation. The infection was detected in the recipient 3 days after transplantation, during the asymptomatic phase. We also report an innovative prophylactic strategy based on infusion of WNV hyperimmune plasma and gamma globulins that could be effective in preventing the appearance of a neuroinvasive disease.