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 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.

The novel anti-inflammatory agent VA694, endowed with both NO-releasing and COX2-selective inhibiting properties, exhibits NO-mediated positive effects on blood pressure, coronary flow and endothelium in an experimental model of hypertension and endothelial dysfunction.

Selective cyclooxygenase 2 (COX2) inhibitors (COXIBs) are effective anti-inflammatory and analgesic drugs with improved gastrointestinal (GI) safety compared to nonselective nonsteroidal anti-inflammatory drugs known as traditional (tNSAIDs). However, their use is associated with a cardiovascular (CV) hazard (i.e. increased incidence of thrombotic events and hypertension) due to the inhibition of COX2-dependent vascular prostacyclin. Aiming to design COX2-selective inhibitors with improved CV safety, new NO-releasing COXIBs (NO-COXIBs) have been developed. In these hybrid drugs, the NO-mediated CV effects are expected to compensate for the COXIB-mediated inhibition of prostacyclin. This study evaluates the potential CV beneficial effects of VA694, a promising NO-COXIB, the anti-inflammatory effects of which have been previously characterized in several in vitro and in vivo experimental models. When incubated in hepatic homogenate, VA694 acted as a slow NO-donor. Moreover, it caused NO-mediated relaxant effects in the vascular smooth muscle. The chronic oral administration of VA694 to young spontaneously hypertensive rats (SHRs) significantly slowed down the age-related development of hypertension and was associated with increased plasma levels of nitrates, stable end-metabolites of NO. Furthermore, a significant improvement of coronary flow and a significant reduction of endothelial dysfunction were observed in SHRs submitted to chronic administration of VA694. In conclusion, VA694 is a promising COX2-inhibiting hybrid drug, showing NO releasing properties which may mitigate the CV deleterious effects associated with the COX2-inhibition.

Fyn kinase in brain diseases and cancer: the search for inhibitors.

Fyn is a non-receptor tyrosine kinase belonging to the Src family kinases. It has been shown to play important roles in neuronal functions, including myelination and oligodendrocytes formation, and in inflammatory processes. It has also demonstrated its involvement in signaling pathways that lead to severe brain pathologies, such as Alzheimer's and Parkinson's diseases. Moreover, Fyn is upregulated in some malignancies. Experimental studies demonstrated that Fyn inhibition could be useful in the disruption of metabolic processes involved in cancer neurodegenerative diseases. Unfortunately, no specific Fyn inhibitor has been discovered till today, active compounds on other members of Src family or on different tyrosine kinases have also been reported. However, multitargeted inhibitors might be endowed with therapeutic potential. Indeed, as increasingly reported, also a not completely selective inhibitor of a specific protein could be therapeutically useful, affecting a number of cell pathways involved especially in cancer development. In this review, we report some examples of small molecule tyrosine kinase inhibitors for which data on Fyn inhibition, both in enzymatic and in cell assays, have been reported, with the aim of giving information as starting point for the researchers working in this field.

Enlarging the NSAIDs family: ether, ester and acid derivatives of the 1,5-diarylpyrrole scaffold as novel anti-inflammatory and analgesic agents.

The development of the coxib family has represented a stimulating approach in the treatment of inflammatory disorders, such as arthritis, and for the management of acute pains, in relation to the well-known traditional Non-Steroidal Anti-inflammatory Drugs (t-NSAIDs). Prompted by the pursuit for new cyclooxygenase-2 (COX-2) inhibitors, endowed with fine tuned selectivity and high potency, in the past years we have identified novel classes of ether, ester and acid molecules characterized by the 1,5-diarylpyrrole scaffold as potentially powerful anti-inflammatory molecules (12-66). All compounds proved to exert an in vitro inhibition profile as good as that shown by reference compounds. Compounds bearing a p-methylsulfonylphenyl substituent at C5 displayed the best issues. In particular, ester derivatives proved to perform the best in vitro profile in terms of selectivity and activity toward COX-2. The cell-based assay data showed that an increase of hindrance at the C3 side chain of compounds could translate to activity enhancement. The human whole blood (HWB) test let to highlight that submitted compounds displayed 5-10 fold higher selectivity for COX-2 vs COX-1 which should translate clinically to an acceptable gastrointestinal safety and mitigate the cardiovascular effects highlighted by highly selective COX-2 inhibitors. Finally, to assess in vivo anti-inflammatory and analgesic activity three different tests (rat paw pressure, rat paw oedema and abdominal constriction) were performed. Results showed good in vivo anti-inflammatory and analgesic activities. The issues gained with these classes of compounds represent, nowadays, a potent stimulus for a further enlargement of the NSAIDs family. In this review we describe the results obtained by our research group on this topic.

New derivatives of BM212: A class of antimycobacterial compounds based on the pyrrole ring as a scaffold.

During our investigation in the area of antimycobacterial agents, we have identified the 1,5-(4-chlorophenyl)-2-methyl-3-(4-methylpiperazin-1-yl)methyl-1H-pyrrole (BM212) as the lead compound for a new class of antimycobacterial pyrrole derivatives with potent in vitro activity against mycobacteria and with low cytotoxicity. We have also identified the salient structural features of BM212, while structure-activity relationships (SAR) and molecular modeling studies on pyrrole compounds allowed us to design and synthesize additional analogues. Among them, seven compounds revealed a very high activity (better than that of BM212 toward mycobacteria) and a very interesting protection index, comparable to that of reference compounds, such as isoniazid, streptomycin and rifampin.

Development of a PCR assay followed by nonradioactive hybridization using oligonucleotides covalently bound to CovaLink NH microwells for detection of four Plasmodium species in blood samples from humans.

We developed and evaluated a PCR-based assay to detect four Plasmodium species in 79 blood samples from 56 travelers returning from areas where malaria is endemic. DNA amplification targeting a small region of the 18S rRNA gene was performed with Plasmodium genus-specific primers. The biotinylated PCR products were then identified by PCR-colorimetric Covalink NH microwell plate hybridization (CMPH) using species-specific phosphorylated probes covalently bound to a pretreated polystyrene surface. The results from PCR-CMPH showed high specificity, and for 47 of the 56 patients (84%), microscopy and PCR-CMPH results were in agreement. Discordant results were reevaluated with microscopy examination, other molecular methods, and DNA sequencing. Except for one patient, discrepancies were resolved in favor of PCR-CMPH: three mixed infections were detected, four species identification errors were corrected, and two negative results were shown to be positive. Our results indicate that PCR-CMPH is a simple, rapid, and specific method for malaria diagnosis. It employs stable reagents and inexpensive equipment, making it suitable for routine epidemiological use.

New trends in development of antimycobacterial compounds.

The resurgence of tuberculosis and the surge of multidrug-resistant clinical isolates of Mycobacterium tuberculosis have reaffirmed tuberculosis as a primary public health concern. In this review we describe some new findings on the pharmacological status of fluoroquinolones derivatives (Gatifloxacin, Moxifloxacin and Sitafloxacin), new macrolides (Clarithromycin, Azithromycin and Roxithromycin), new rifamycin derivatives (Rifapentin, Rifabutin and Rifalazil) and new oxazolidinones (Linezolid and PNU 100480). We describe also other type of agents that are being developed as antimycobacterial drugs. Some of these are under clinical investigation, while others are considered to be promising candidates for future development. Among them, nitroimidazopyrans, new ketolides, Isoxyl (ISO), pyrroles derived from BM 212, Mefloquine and Diarylquinoline R207910 are discussed. We also describe the mechanism of drug resistance in mycobacteria, as well as new potential targets.

Genetic identification of the main opportunistic Mucorales by PCR-restriction fragment length polymorphism.

Mucormycosis is a rare and opportunistic infection caused by fungi belonging to the order Mucorales. Recent reports have demonstrated an increasing incidence of mucormycosis, which is frequently lethal, especially in patients suffering from severe underlying conditions such as immunodeficiency. In addition, even though conventional mycology and histopathology assays allow for the identification of Mucorales, they often fail in offering a species-specific diagnosis. Due to the lack of other laboratory tests, a precise identification of these molds is thus notoriously difficult. In this study we aimed to develop a molecular biology tool to identify the main Mucorales involved in human pathology. A PCR strategy selectively amplifies genomic DNA from molds belonging to the genera Absidia, Mucor, Rhizopus, and Rhizomucor, excluding human DNA and DNA from other filamentous fungi and yeasts. A subsequent digestion step identified the Mucorales at genus and species level. This technique was validated using both fungal cultures and retrospective analyses of clinical samples. By enabling a rapid and precise identification of Mucorales strains in infected patients, this PCR-restriction fragment length polymorphism-based method should help clinicians to decide on the appropriate treatment, consequently decreasing the mortality of mucormycosis.

Laboratory diagnosis of cystic hydatic disease.

The main purpose of this article is to answer the questions about which test to perform for hydatic diagnosis and when. Several techniques for biologic diagnosis and follow-up of human cystic hydatidosis are reviewed. The specificity and sensitivity of immunologic reactions are reported. The differential diagnosis between Echinococcus granulosus and E. multilocularis is examined. The characteristics of the immunologic diagnosis according to the stage and the treatment of hydatidosis disease is discussed. Laboratory diagnosis of cystic hydatic disease is complementary to the clinical data. A judicious association of the usual techniques (indirect immunofluorescence assay, indirect hemagglutination assay, immunoelectrophoresis, co-electrophoresis with antigen 5 identification) confirms the diagnosis in 80% to 94% of hepatic hydatidosis cases and in 65% of pulmonary hydatidosis cases. Special techniques (enzyme-linked immunosorbent assay, Western blot, polymerase chain reaction) must be used for other localizations or when cysts are calcified. A serologic survey is necessary for the follow-up of operated medically treated patients. Despite poor standardization, purified antigens can distinguish between E. granulosus and E. multilocularis infections, although false-positive results are observed during other helminthiases, such as cysticerocosis.

Building a pharmacophore model for a novel class of antitubercular compounds.

Starting from a set of 32 antitubercular compounds, for the first time a three-dimensional pharmacophore model has been derived through a computational approach based on CATALYST software. The model proved to be able to identify compounds belonging to classes of molecules already reported as antitubercular agents.

New pyrrole derivatives as antimycobacterial agents analogs of BM212.

During the course of our investigations in the field of azole antimicrobial agents, we have identified BM212 a pyrrole derivative with good in vitro activity against mycobacteria and candidae. These findings prompted us to prepare new pyrrole derivatives 2-6 in the hope of increasing the activity. The microbiological data showed interesting in vitro activity against Mycobacterium tuberculosis and atypical mycobacteria.

Autoimmune events during interferon beta-1b treatment for multiple sclerosis.

Autoimmune events, although rarely reported during interferon beta-1b (IFNB) treatment of relapsing-remitting (RR) multiple sclerosis (MS), may be more frequent than expected due to the many immunologic abnormalities associated with this disease. We report the prospective two-year follow-up of autoimmune events in 40 RR MS patients treated with IFNB and in 21 untreated MS controls. Thyroid and liver function and serum level of 12 autoantibodies (autoAbs) against organ- (thyroid, gastric, pancreatic) and non-organ-specific antigens were serially monitored. In contrast to control patients, autoAbs (anti-nuclear, -smooth muscle or -thyroid antigens) were detected in 13 IFNB-treated patients, and these were associated with thyroid or liver function alteration in many cases. Persistent autoimmune thyroid dysfunction occurred in three IFNB-treated patients, all of whom were women with a familial history of thyroid disease or baseline anti-thyroid autoAb positivity. For improvement of the MS relapse rate, thyroid dysfunction was adequately treated without stopping IFNB. Liver function alteration (17 IFNB-treated patients, associated with non-organ-specific autoAbs in four) was transient and did not require IFNB treatment to be stopped, with the exception of one patient who was already suffering from a drug-induced hepatopathy at baseline. During the IFNB treatment of MS, several autoimmune events may occur, indicating that thyroid and liver function and autoAbs must be carefully monitored.

Role of coinfection by transfusion-transmitted virus (TTV) in patients with chronic HCV hepatitis.

A novel DNA virus, associated with non A-G post-transfusion hepatitis, has been recently discovered. TTV is detectable in acute and chronic liver diseases with a variable incidence. The clinical significance of TTV coinfection in patients with chronic HCV hepatitis has not yet been defined. In this review we report the data regarding the possible pathogenetic role of TTV infection in chronic HCV hepatitis.

Antimycobacterial activity of new ortho-, meta- and para-toluidine derivatives.

Novel toluidine derivatives are described. Some of them showed an interesting in vitro activity against Mycobacterium tuberculosis, M. smegmatis, M. marinum, M. gordonae, and M. avium. Some of them were more active than Streptomycin and Isoniazid, which were used as controls, against M. avium and M. gordonae. In particular, we confirm the good activity of biphenyl derivatives.

Bactericidal activities of the pyrrole derivative BM212 against multidrug-resistant and intramacrophagic Mycobacterium tuberculosis strains.

The pyrrole derivative BM212 [1, 5-diaryl-2-methyl-3-(4-methylpiperazin-1-yl)methyl-pyrrole] was shown to possess strong inhibitory activity against both Mycobacterium tuberculosis and some nontuberculosis mycobacteria. BM212 was inhibitory to drug-resistant mycobacteria and also exerted bactericidal activity against intracellular bacilli residing in the U937 human histiocytic lymphoma cell line.

[HCV and extrahepatic diseases]. / HCV e patologie extraepatiche.

Besides being a hepatotropic virus and a common cause of chronic hepatitis, hepatitis C virus (HCV) has been linked to a variety of extrahepatic immunological manifestations. The high prevalence of HCV infections in some of these conditions suggests an important pathogenetic role of the virus. The recent observation that HCV infects peripheral blood mononuclear cells, such as CD8+ T lymphocytes, CD19+ B lymphocytes and monocytes/microphages, has given an insight into the possible mechanisms of HCV associated autoimmunity. In the clinical practice it is recommended not only to search for symptoms and signs of autoimmune disorders in patients with chronic hepatitis C, but also to test for hepatitis C virus infection patients with extrahepatic conditions known to be related to HCV. Even if the occurrence of autoimmune disorders or the exacerbations of autoimmune diseases has been reported during interferon therapy, antiviral therapy is effective in treating some of the extrahepatic disorders associated with HCV, namely mixed cryoglobulinemia and membranoproliferative glomerulonephritis. The extrahepatic manifestations associated with hepatitis C virus infection are reviewed according to the available data and the option of interferon therapy is discussed.

[LKM (Liver-Kidney-Microsome) antibodies and hepatitis C virus. Description of a clinical case]. / Anticorpi LKM (Liver-Kidney Microsome) e virus C. Descrizione di un caso clinico.

BACKGROUND: Diagnostic and therapeutic issues related to hepatitis C virus infection and autoimmune hepatitis are discussed. The authors report a 56 year old female patient with chronic hepatitis and both HCV-RNA positivity and a high titer of LKM-1 antibody on blood samples. METHODS: In the absence of clinical signs of autoimmunity the patient was started on interferon treatment. After four months she experienced a flare-up with a sharp increase of transaminases and a concomitant rise in LKM-1 titer. Viremia was persistently detected by PCR. As interferon therapy was discontinued transaminases and autoantibody titer fell to baseline values. A few months later she received immunosuppressive therapy, that resulted in a decrease in LKM1 titer and complete normalization of liver enzymes. Anti LKM-1 antibody was detected by indirect immunofluorescence, serum immunoblot assay (Western Blot), and enzyme immunoassay (ELISA). RESULTS AND CONCLUSIONS: Therapy of patients with HCV/LKM positive chronic hepatitis should be settled on an individual basis. Patients eligible for interferon treatment should be carefully selected and closely monitored because of the risk of adverse reaction.

Toxoplasmosis in kidney transplant recipients: report of six cases and review.

Six patients with toxoplasmosis complicating renal transplantation are described, and 25 other reported cases are reviewed. The mean age of the 31 patients was 35.16 years. Most of the recipients (25 of 29) showed signs of toxoplasmosis within 3 months post-transplantation, with fever, neurological disturbances, and pneumonia as the main clinical features. Diagnosis was established at autopsy in 15 cases, by serology in 13 cases, and by direct examination, culture, or polymerase chain reaction of biological samples in 5 cases. Seventeen patients also had concomitant infections. The donor was the likely source of transmission to 10 recipients; reactivation was suspected in two cases. The source of transmission could not be determined for the remaining 19 patients. The mortality rate was 64.5%. Ten of the 11 patients given specific treatment survived, indicating that early diagnosis and therapy are essential.