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.

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.

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.

Effect of nomegestrol acetate on estrogen biosynthesis and transformation in MCF-7 and T47-D breast cancer cells.

Although ovaries serve as the primary source of estrogen for pre-menopausal women, after menopause estrogen biosynthesis from circulating precursors occurs in peripheral tissues by the action of several enzymes, 17beta-hydroxysteroid dehydrogenase 1 (17beta-HSD1), aromatase and estrogen sulfatase. In the breast, both normal and tumoral tissues have been shown to be capable of synthesizing estrogens, and this local estrogen production can be implicated in the development of breast tumors. In these tissues, estradiol (E(2)) can be synthesized by three pathways: (1) estrone sulfatase transforms estrogen sulfates into bioactive estrogens, (2) 17beta-HSD1 converts estrone (E(1)) into E(2), (3) aromatase which converts androgens into estrogens is also present and contributes to the in situ synthesis of active estrogens but to a far lesser extent than estrone sulfatase. Quantitative assessment of E(2) formation in human breast tumors indicates that metabolism of estrone sulfate (E(1)S) via the sulfatase pathway produces 100-500 times more E(2) than androgen aromatization. Breast tissue also possesses the estrogen sulfotransferase involved in the conversion of estrogens into their sulfates that are biologically inactive. In the present review, we summarized the action of the 19-nor-progestin nomegestrol acetate (NOMAC) on the sulfatase, 17beta-HSD1 and sulfotransferase activities in the hormone-dependent MCF-7 and T47-D human breast cancer cell lines. Using physiological doses of substrates NOMAC blocks very significantly the conversion of E(1)S to E(2). It inhibits the transformation of E(1) to E(2). NOMAC has a stimulatory effect on sulfotransferase activity in both cell lines, with a strong stimulating effect at low doses but only a weak effect at high concentrations. The effects on the three enzymes are always stronger in the progesterone-receptor rich T47-D cell line as compared with the MCF-7 cell line. Besides, no effect is found for NOMAC on the transformation of androstenedione to E(1) in the aromatase-rich choriocarcinoma cell line JEG-3. In conclusion, the inhibitory effect provoked by NOMAC on the enzymes involved in the biosynthesis of E(2) (sulfatase and 17HSD pathways) in estrogen-dependent breast cancer, as well as the stimulatory effect on the formation of the inactive E(1)S, can open attractive perspectives for future clinical trials.

In vitro and in vivo models for the evaluation of new inhibitors of human steroid sulfatase, devoid of residual estrogenic activity.

The goal of our research project is to develop a new class of orally active drugs, estrone sulfatase inhibitors, for the treatment of estrogen-dependent (receptor positive) breast cancer. Several compounds were synthesized and their pharmacological potencies explored. Based on encouraging preliminary results, three of them, TX 1299, TX 1492 and TX 1506 were further studied in vitro as well as in vivo. They proved to be strong inhibitors of estrone sulfatase when measured on the whole human JEG-3 choriocarcinoma and MCF-7 breast cancer cells and their IC(50)s found to be in the range of known standard inhibitors. Their residual estrogenic activity was checked as negative in the test of induction of alkaline phosphatase (APase) activity in whole human endometrial adenocarcinoma Ishikawa cells. In addition, their effect on aromatase activity in JEG-3 cells was also examined, since the goal of inhibiting both sulfatase and aromatase activities appears very attractive. However, it has been unsuccessful so far. Then, in vivo potencies of TX 1299, the lead compound in our chemical series, were evaluated in comparison with 6,6,7-COUMATE, a non-steroidal standard, in two different rat models and by oral route. First, the absence of any residual estrogenic activity for these compounds was checked in the uterotrophic model in prepubescent female rats. Second, antiuterotrophic activity in adult ovariectomized rat supplemented with estrone sulfate (E(1)S), showed that both compounds were potent inhibitors, the power of TX 1299 relative to 6,6,7-COUMATE being around 80%. This assay was combined with uterine sulfatase level determination and confirmed the complete inhibition of this enzyme within the target organ. Preliminary studies indicated that other non-steroid compounds in the Théramex series were potent in vitro and in vivo inhibitors of estrone sulfatase in rats and further studies are in progress.