Exploring MALDI-TOF MS approach for a rapid identification of Mycobacterium avium ssp. paratuberculosis field isolates.

AIMS: The aim of the study was to explore the suitability of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and correct identification of Mycobacterium avium ssp. paratuberculosis (MAP) field isolates. METHODS AND RESULTS: MALDI-TOF MS approach is becoming one of the most popular tests for the identification of intact bacterial cells which has been shown to be fast and reliable. For this purpose, 36 MAP field isolates were analysed through MALDI-TOF MS and the spectra compared with two different databases: one provided by the vendor of the system employed (Biotyper ver. 3·0; Bruker Daltonics) and a homemade database containing spectra from both tuberculous and nontuberculous Mycobacteria. Moreover, principal component analysis procedure was employed to confirm the ability of MALDI-TOF MS to discriminate between very closely related subspecies. Our results suggest MAP can be differentiated from other Mycobacterium species, both when the species are very close (M. intracellulare) and when belonging to different subspecies (M. avium ssp. avium and M. avium ssp. silvaticum). CONCLUSIONS: The procedure applied is fast, easy to perform, and achieves an earlier accurate species identification of MAP and nontuberculous Mycobacteria in comparison to other procedures. SIGNIFICANCE AND IMPACT OF THE STUDY: The gold standard test for the diagnosis of paratuberculosis is still isolation of MAP by cultural methods, but additional assays, such as qPCR and subculturing for determination of mycobactin dependency are required to confirm its identification. We have provided here evidence pertaining to the usefulness of MALDI-TOF MS approach for a rapid identification of this mycobacterium among other members of M. avium complex.

Survey of laboratory-acquired infections around the world in biosafety level 3 and 4 laboratories.

Laboratory-acquired infections due to a variety of bacteria, viruses, parasites, and fungi have been described over the last century, and laboratory workers are at risk of exposure to these infectious agents. However, reporting laboratory-associated infections has been largely voluntary, and there is no way to determine the real number of people involved or to know the precise risks for workers. In this study, an international survey based on volunteering was conducted in biosafety level 3 and 4 laboratories to determine the number of laboratory-acquired infections and the possible underlying causes of these contaminations. The analysis of the survey reveals that laboratory-acquired infections have been infrequent and even rare in recent years, and human errors represent a very high percentage of the cases. Today, most risks from biological hazards can be reduced through the use of appropriate procedures and techniques, containment devices and facilities, and the training of personnel.

Management of pregnant women infected with Ebola virus in a treatment centre in Guinea, June 2014.

We report two cases of confirmed Ebola virus disease in pregnant women, who presented at the Médecins Sans Frontières Ebola treatment centre in Guéckédou. Despite the very high risk of death, both pregnant women survived. In both cases the critical decision was made to induce vaginal delivery. We raise a number of considerations regarding the management of Ebola virus-infected pregnant women, including the place of amniocentesis and induced delivery, and whether certain invasive medical acts are justified.

The prevalence of antibodies to human herpesvirus 8 and hepatitis B virus in patients in two hospitals in Tanzania.

The aim of this study was to determine the seroprevalence of human herpesvirus 8 (HHV-8) and the immunization status for hepatitis B virus (HBV) infection in febrile patients in two districts of the United Republic of Tanzania. Between February and March 2007, blood samples were collected in Pemba Island and Tosamaganga from 336 outpatients and sent to the Virology Laboratory in Rome (Italy) for testing. HHV-8 DNA and HBV-DNA were amplified by two in-house molecular methods, anti-HHV-8 antibody titers were determined by an immunofluorescence assay (IFA), and anti-HCV, HBsAg, anti-HBs, and anti-HBc were evaluated by microplate enzyme immunoassay (MEIA). The seroprevalence of HHV-8 was 30.7% (96/313). In Pemba Island, the prevalence was lower than in Tosamaganga (14.4% vs. 46.3%). A higher prevalence of low titers of HHV-8 IgG (<1:80, 81%) was found among those under 5 years of age. HHV-8 DNA was detected in six seropositive patients (6.7%). The prevalence of HBsAg, anti-HBs, and anti-HBc was 4.3%, 37.6%, and 29.3%, respectively. Out of 277 patients, 70 had had a previous infection (25.3%). One case of occult hepatitis was found. The cover of hepatitis B vaccination was higher among children born after 2002 (66.7%) than in patients born before 2002. HHV-8 infection is endemic in Tanzania and the seroprevalence rate was higher in the mainland than on Pemba Island. The 3.9% percentage of HBsAg in children younger than 4 years of age suggests that increased efforts are required in order to achieve universal and compulsory immunization of children against HBV.

Recent expansion of dengue virus serotype 3 in West Africa.

Due to non-existing or limited surveillance in Africa, little is known about the epidemiology of dengue illness in the continent. Serological and virological data obtained from returning European travellers is a key complement to this often flawed information. In the past years, dengue 3 virus has emerged in West Africa and has been detected in travellers returning to Europe. The first dengue epidemic in Cape Verde with more than 17,000 cases from September to December 2009 demonstrated that dengue virus is still expanding worldwide to new territories.

A case of dengue type 3 virus infection imported from Africa to Italy, October 2009.

In October 2009, a traveller returning from Africa to Italy was hospitalised with symptoms suggestive of a haemorrhagic fever of unknown origin. The patient was immediately placed in a special biocontainment unit until laboratory investigations confirmed the infection to be caused by a dengue serotype 3 virus. This case reasserts the importance of returning travellers as sentinels of unknown outbreaks occurring in other countries, and highlights how the initial symptoms of dengue fever resemble those of other haemorrhagic fevers, hence the importance of prompt isolation of patients until a final diagnosis is reached.

HIV-particles in spermatozoa of patients with AIDS and their transfer into the oocyte.

By immunocytochemistry and in situ hybridization at the electron microscopy level, and by the PCR technique, we have shown that HIV-1 binds and enters normal sperm; that viral particles, their antigens, and nucleic acid are present in sperm from HIV-1 infected men; and that such sperm can transfer HIV-1 like particles to normal human oocytes. We also present evidence that a galactosylceramide-like compound is present on the sperm membrane and could function as an alternative receptor for HIV.

Multidrug-Resistant Tuberculosis In A Referral Center In Rome: 2011- 2016.

PURPOSE: Multidrug-resistant tuberculosis (MDR-TB) is a major burden to public health in low incidence countries in Europe. The aim of this study was to attempt to have a better insight into the trends of MDR-TB in the metropolitan area of Rome, within the Italian and the foreign-born population, based on microbiological and demographic data. PATIENTS AND METHODS: We performed a prospective study, collecting microbiological data based on phenotypic drug-resistant testing (DST) of TB strains consecutively isolated in a referral hospital in Rome, the capital city of a low TB incidence country, over a 6-year period, and correlated them to the geographical origin of patients. This study was carried out in a referral hospital for patients with drug-resistant TB from the whole region. RESULTS: Drug-resistance data from 926 patients with a microbiological diagnosis of TB from 2011 to 2016 show a 5.5% rate of MDR-TB, mostly occurring in patients born in a single East European country, that has a high incidence of MDR-TB. The strains isolated from these patients frequently carry additional resistances, leading to an increased risk of developing extensively drug-resistant (XDR) TB. CONCLUSION: In the great metropolitan area of Rome, MDR-TB more frequently occurs in patients who were born in a single country from Eastern Europe known to have high rates of MDR-TB and long-time residents in Italy. Recent immigrants from non-European countries do not appear to contribute to the rates of MDR-TB reported in this article. This knowledge of local TB trends could help improve the measures of surveillance and prevention of disease.

Clinical evaluation of TRCReady M.TB for rapid automated detection of M. tuberculosis complex in respiratory samples.

SETTING: Timely diagnosis of tuberculosis (TB) is essential for effectively controlling and managing the disease. Although international guidelines recommend acid-fast bacilli staining and culture as the 'gold standard', new molecular methods are available to safely and rapidly identify positive samples. OBJECTIVE: To evaluate the performance of the newer and fully automated version of a molecular assay for rRNA amplification (TRCReady® M.TB) on 1028 respiratory samples collected from 378 patients for its possible use as a reliable screening method. Results were evaluated using culture as the reference test. RESULTS: Of four diagnostic protocols employed, best results were obtained when TRCReady M.TB was used together with microscopy on the first respiratory sample, followed by microscopy alone on a second one. The sensitivity and specificity were respectively 97% and 100%, with a turnaround time of 24 h. We propose a possible laboratory algorithm for rapid identification of patients with TB. CONCLUSIONS: TRCReady offers the advantages of full automation and avoidance of cross-contamination. As such, it should be considered as a more economical option for TB screening than other commercial assays that are currently available.

Colonization and infection due to carbapenemase-producing Enterobacteriaceae in liver and lung transplant recipients and donor-derived transmission: a prospective cohort study conducted in Italy.

OBJECTIVES: A prospective cohort study was conducted in Italy in order to describe the microbiologic aspects of colonization/infection by carbapenemase-producing Enterobacteriaceae (CPE) in donors and recipients of lung and liver transplants and the possible CPE transmission from donors to recipients. METHODS: Between 15 January 2014 and 14 January 2015, all recipients of solid organ transplants (SOT) at ten lung and eight liver transplantation centres and the corresponding donors were enrolled. Screening cultures to detect CPE were performed in donors, and screening and clinical cultures in recipients with a 28-day microbiologic follow-up after receipt of SOT. Detection of carbapenemase genes by PCR, genotyping by multilocus sequence typing, and pulsed-field gel electrophoresis and whole-genome sequencing were performed. RESULTS: Of 588 screened donors, 3.4% were colonized with CPE. Of the liver first transplant recipients (n = 521), 2.5% were colonized before receipt of SOT and 5% acquired CPE during follow-up. CPE colonization was higher in lung first transplant recipients (n = 111, 2.7% before SOT and 14.4% after SOT). CPE infections occurred in 1.9% and 5.3% of liver or lung recipients, respectively. CPE isolates were mostly Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae belonging to CG258. Three events of donor-recipient CPE transmission, confirmed by whole-genome sequencing and/or pulsed-field gel electrophoresis, occurred in lung recipients: two involving K. pneumoniae sequence type 512 and one Verona integron-encoded metallo-ß-lactamase (VIM)-producing Enterobacter aerogenes. CONCLUSIONS: This study showed a low risk of donor-recipient CPE transmission, indicating that donor CPE colonization does not necessarily represent a contraindication for donation unless colonization regards the organ to be transplanted. Donor and recipient screening remains essential to prevent CPE transmission and cross-infection in transplantation centres.

Occupational transmission of an Orthopoxvirus infection during an outbreak in a colony of Macaca tonkeana in Lazio Region, Italy, 2015.

Orthopoxviruses spill over from animal reservoirs to accidental hosts, sometimes causing human infections. We describe the surveillance and infection control measures undertaken during an outbreak due to an Orthopoxvirus occurred in January 2015 in a colony of Macaca tonkeana in the province of Rieti, Latio, Italy, which caused a human asymptomatic infection. According to the epidemiological investigation, the human transmission occurred after an unprotected exposure. The contacts among wild, captive and domestic animals and humans, together with decreased immunity against Orthopoxviruses in the community, may put animal handlers at risk of infection, especially after the cessation of smallpox vaccination. To reduce these threats, standard precautions including respiratory hygiene and transmission-based precautions should be carefully applied also in veterinary medicine.

EBOLA Ag K-SeT rapid test: field evaluation in Sierra Leone.

OBJECTIVES: Efficient interruption of Ebola virus disease (EVD) transmission chains critically depends on reliable and fast laboratory diagnosis. We evaluated the performance of the EBOLA Virus Antigen Detection K-SeT (EBOLA Ag K-SeT), a new rapid diagnostic antigen test in field settings. METHODS: The study was conducted in a field laboratory located in Freetown (Sierra Leone) by the Italian National Institute for Infectious Diseases 'L. Spallanzani' and the EMERGENCY Onlus NGO. The EBOLA Ag K-SeT was tested on 210 residual plasma samples (EVD prevalence 50%) from patients hospitalized at the EMERGENCY Ebola treatment center in Goderich (Freetown), comparing the results with quantitative real-time PCR. RESULTS: Overall, the sensitivity of EBOLA Ag K-SeT was 88.6% (95% confidence interval (CI), 82.5-94.7), and the corresponding specificity was 98.1% (95% CI, 95.5-100.7). The positive and negative predictive values were 97.9% (95% CI, 95.0-100.8) and 89.6% (95% CI, 84-95.2), respectively. The sensitivity strongly increased up to 98.7% (95% CI, 96.1-101.2) for those samples with high virus load (≥6.2 log RNA copies/mL). CONCLUSIONS: Our results suggest that EBOLA Ag K-SeT could represent a new effective diagnostic tool for EVD, meeting a need for resource-poor settings and rapid diagnosis for individuals with suspected EVD.

Evaluation of a rapid and sensitive RT-qPCR assay for the detection of Ebola Virus.

BACKGROUND: The 2013-2016 Ebola virus disease (EVD) outbreak showed a lack of diagnostic point-of-care methods. Currently, EBOV diagnosis relies on quantitative reverse-transcription-PCR (RT- qPCR), highly specific and sensitive, but requiring skilled personnel and well-equipped laboratories. In field settings, these factors and others, such as samples' time of collection and transportation, determine a prolonged turnaround-time to final results. In outbreak scenarios, a rapid and transportable method could eliminate issues of cohorting suspected and actual EVD patients for lack of diagnostic certainty. The aim of this study was the field evaluation of the new fast, easy-to-use and reliable RT-qPCR assay and platform for EBOV detection, developed in the framework of the EbolaMoDRAD project by CLONIT S.r.l. and STMicroelectronics S.r.l. STUDY DESIGN: We evaluated its performance during the outbreak and in further studies in the EVD laboratory at the Princess Christian Maternity Hospital (PCMH) in Freetown (Sierra Leone) run by Emergency NGO and the Italian National Institute for Infectious Diseases (INMI). The assay was tested on residual aliquots of clinical specimens from EBOV-positive or -negative patients (n=116, EVD prevalence 37%). RESULTS AND CONCLUSION: Overall, the test was very easy-to-use and the instrument was robust and reliable in field-settings. The sensitivity of the assay was 100% and the specificity was 98.63% (95%CI: 96.34-100.92%). The positive and negative predictive values were 97.73 (95%CI:94.77-100.68%) and 100%, respectively. The high sensitivity and specificity of this new assay indicate that it is promising for laboratory diagnosis, especially in resource-limited settings.

Ebola virus infection induces autoimmunity against dsDNA and HSP60.

Ebola virus (EBOV) survivors are affected by a variety of serious illnesses of unknown origin for years after viral clearance from the circulation. Identifying the causes of these persistent illnesses is paramount to develop appropriate therapeutic protocols. In this study, using mouse and non-human primates which survived EBOV challenge, ELISA, western blot, mass spectrometry and flow cytometry were used to screen for autoantibodies, identify their main targets, investigate the mechanism behind their induction and monitor autoantibodies accumulation in various tissues. In infected mice and NHP, polyclonal B cell activation and autoantigens secretion induced autoantibodies against dsDNA and heat shock protein 60 as well as antibody accumulation in tissues associated with long-term clinical manifestations in humans. Finally, the presence of these autoantibodies was confirmed in human EBOV survivors. Overall, this study supports the concept that autoimmunity is a causative parameter that contributes to the various illnesses observed in EBOV survivors.

Longitudinal characterization of dysfunctional T cell-activation during human acute Ebola infection.

Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells.

Hospital preparedness and management of patients affected by viral haemorrhagic fever or smallpox at the Lazzaro Spallanzani Institute, Italy.

The US cases of anthrax in 2001 and the recent severe acute respiratory syndrome outbreak have heightened the need for preparedness and response to naturally emerging and re-emerging infections or deliberately released biological agents. This report describes the response model of the Istituto Nazionale per le Malattie Infettive Lazzaro Spallanzani (INMI), Rome, Italy for managing patients suspected of or affected by smallpox or viral haemorrhagic fever (VHF) either in the context of an intentional release or natural occurrence. The INMI is Italy's leading hospital in its preparedness and response plan to bioterrorism-related infectious agents. All single and double rooms of INMI are equipped with negative air pressure, sealed doors, high efficiency particulate air (HEPA) filters and a fully-equipped anteroom; moreover, a dedicated high isolation unit with a laboratory next door for the initial diagnostic assays is available for admission of sporadic patients requiring high isolation. For patient transportation, two fully equipped ambulances and two stretcher isolators with a negative pressure section are available. Biomolecular and traditional diagnostic assays are currently performed in the biosafety level 3/4 (BSL 3/4) laboratories. Continuing education and training of hospital staff, consistent application of infection control practices, and availability of adequate personnel protective equipment are additional resources implemented for the care of highly infectious patients and to maintain the readiness of an appropriately trained workforce to handle large scale outbreaks.

Hospital preparedness and management of patients affected by viral haemorrhagic fever or smallpox at the Lazzaro Spallanzani Institute, Italy.

The US cases of anthrax in 2001 and the recent severe acute respiratory syndrome outbreak have heightened the need for preparedness and response to naturally emerging and re-emerging infections or deliberately released biological agents. This report describes the response model of the Istituto Nazionale per le Malattie Infettive Lazzaro Spallanzani (INMI), Rome, Italy for managing patients suspected of or affected by smallpox or viral haemorrhagic fever (VHF) either in the context of an intentional release or natural occurrence. The INMI is Italy's leading hospital in its preparedness and response plan to bioterrorism-related infectious agents. All single and double rooms of INMI are equipped with negative air pressure, sealed doors, high efficiency particulate air (HEPA) filters and a fully-equipped anteroom; moreover, a dedicated high isolation unit with a laboratory next door for the initial diagnostic assays is available for admission of sporadic patients requiring high isolation. For patient transportation, two fully equipped ambulances and two stretcher isolators with a negative pressure section are available. Biomolecular and traditional diagnostic assays are currently performed in the biosafety level 3/4 (BSL 3/4) laboratories. Continuing education and training of hospital staff, consistent application of infection control practices, and availability of adequate personnel protective equipment are additional resources implemented for the care of highly infectious patients and to maintain the readiness of an appropriately trained workforce to handle large scale outbreaks.