Wolbachia surface protein induces innate immune responses in mosquito cells

BACKGROUND: Wolbachia endosymbiotic bacteria are capable of inducing chronic upregulation of insect immune genes in some situations and this phenotype may influence the transmission of important insect-borne pathogens. However the molecules involved in these interactions have not been characterized. RESULTS: Here we show that recombinant Wolbachia Surface Protein (WSP) stimulates increased transcription of immune genes in mosquito cells derived from the mosquito Anopheles gambiae, which is naturally uninfected with Wolbachia; at least two of the upregulated genes, TEP1 and APL1, are known to be important in Plasmodium killing in this species. When cells from Aedes albopictus, which is naturally Wolbachia-infected, were challenged with WSP lower levels of upregulation were observed than for the An. gambiae cells. CONCLUSIONS: We have found that WSP is a strong immune elicitor in a naturally Wolbachia-uninfected mosquito species (Anopheles gambiae) while a milder elicitor in a naturally-infected species (Aedes albopictus). Since the WSP of a mosquito non-native (nematode) Wolbachia strain was used, these data suggest that there is a generalized tolerance to WSP in Ae. albopictus

Comparative genomics of closely related strains of Klebsiella pneumoniae reveals genes possibly involved in colistin resistance

Strains of colistin-resistant Klebsiella pneumoniae are emerging worldwide, due to the increased use of this molecule in antibiotic-resistant nosocomial infections. Comparative genomics was performed on three closely related K. pneumoniae strains isolated from three patients in a single hospital in Bologna, Italy. Two of these isolates are colistin-resistant, while the third is sensitive to this antibiotic. The designed bioinformatic approach detected, among the three analyzed genomes, single nucleotide polymorphisms, insertions and deletions, specific patterns of gene presence and absence, in a total of 270 genes. These genes were analyzed by automatic and manual methods, to identify those potentially involved in colistin resistance, based on the data available in the literature and on the mechanism of action of colistin, the alteration of the outer membrane. Three of the identified genes (waaL, rfbA, vacJ), all presenting non-synonymous substitutions in the colistin resistant strains, resulted to be of special interest, due to the specific function of their protein products, involved in the biosynthesis of the outer bacterial membrane

Shortage of Albendazole and Its Consequences for Patients with Cystic Echinococcosis Treated at a Referral Center in Italy

Albendazole (ABZ) is the best drug available to treat cystic echinococcosis (CE), a neglected tropical disease. Cystic echinococcosis patients often receive a continuous course of the drug for 6-12 months. In Italy, ABZ shortages occur almost on a yearly basis. We searched clinical records at the World Health Organization Collaborating Center for the Clinical Management of CE in Pavia, Italy, to estimate the amount of ABZ prescribed to patients between January 2012 and February 2017. The cost of ABZ was estimated at €2.25 per tablet based on the current market price in Italy. Patients to whom ABZ had been prescribed were contacted to determine if they had experienced difficulties in purchasing the drug and to assess how such problems affected their treatment. Of 348 identified CE patients, 127 (36.5%) were treated with ABZ for a total of 20,576 days. This led to an estimated cost of €92,592. Seventy-five patients were available for follow-up, 42 (56%) reported difficulties in obtaining ABZ. Of these patients, four (9.5%) had to search out of their region and 10 (23.8%) had to go out of the country. A total of 27 patients (64%) had to visit more than five pharmacies to locate the drug and 10 patients (23.8%) interrupted treatment because of ABZ nonavailability. Shortages in ABZ distribution can disrupt CE treatment schedules and jeopardize patient health

Extracellular non-coding rna signatures of the metacestode stage of echinococcus multilocularis

Extracellular RNAs (ex-RNAs) are secreted by cells through different means that may involve association with proteins, lipoproteins or extracellular vesicles (EV). In the context of parasitism, ex-RNAs represent new and exciting communication intermediaries with promis-ing potential as novel biomarkers. In the last years, it was shown that helminth parasites secrete ex-RNAs, however, most work mainly focused on RNA secretion mediated by EV. Ex-RNA study is of special interest in those helminth infections that still lack biomarkers for early and/or follow-up diagnosis, such as echinococcosis, a neglected zoonotic disease caused by cestodes of the genus Echinococcus. In this work, we have characterised the ex-RNA profile secreted by in vitro grown metacestodes of Echinococcus multilocularis, the casuative agent of alveolar echinococcosis. We have used high throughput RNA-sequencing together with RT-qPCR to characterise the ex-RNA profile secreted towards the extra-and intra-parasite milieus in EV-enriched and EV-depleted fractions. We show that a polarized secretion of small RNAs takes place, with microRNAs mainly secreted to the extra-parasite milieu and rRNA-and tRNA-derived sequences mostly secreted to the intra-parasite milieu. In addition, we show by nanoparticle tracking analyses that viable metacestodes secrete EV mainly into the metacestode inner vesicular fluid (MVF); however, the number of nanoparticles in culture medium and MVF increases > 10-fold when metacestodes show signs of tegument impairment. Interestingly, we confirm the presence of host miRNAs in the intra-parasite milieu, implying their internali-zation and transport through the tegument towards the MVF. Finally, our assessment of the detection of Echinococcus miRNAs in patient samples by RT-qPCR yielded negative results suggesting the tested miRNAs may not be good biomarkers for this disease. A comprehensive study of the secretion mechanisms throughout the life cycle of these parasites will help to understand parasite interaction with the host and also, improve current diagnostic tools.Fil: Ancarola, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Lichtenstein, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Herbig, Johannes. Universität Würzburg; AlemaniaFil: Holroyd, Nancy. No especifíca;Fil: Mariconti, Mara. San Matteo Hospital Foundation; ItaliaFil: Brunetti, Enrico. San Matteo Hospital Foundation; ItaliaFil: Berriman, Matthew. No especifíca;Fil: Albrecht, Krystyna. Universität Würzburg; AlemaniaFil: Marcilla, Antonio. Universidad de Valencia; EspañaFil: Rosenzvit, Mara Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Kamenetzky, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Brehm, Klaus. Universität Würzburg; AlemaniaFil: Cucher, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentin

Comparison of the diagnostic accuracy of three rapid tests for the serodiagnosis of hepatic cystic echinococcosis in humans

BACKGROUND: The diagnosis of cystic echinococcosis (CE) is based primarily on imaging, in particular with ultrasound for abdominal CE, complemented by serology when imaging results are unclear. In rural endemic areas, where expertise in ultrasound may be scant and conventional serology techniques are unavailable due to lack of laboratory equipment, Rapid Diagnostic Tests (RDTs) are appealing. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the diagnostic accuracy of 3 commercial RDTs for the diagnosis of hepatic CE. Sera from 59 patients with single hepatic CE cysts in well-defined ultrasound stages (gold standard) and 25 patients with non-parasitic cysts were analyzed by RDTs VIRapid HYDATIDOSIS (Vircell, Spain), Echinococcus DIGFA (Unibiotest, China), ADAMU-CE (ICST, Japan), and by RIDASCREEN Echinococcus IgG ELISA (R-Biopharm, Germany). Sensitivity, specificity and ROC curves were compared with McNemar and t-test. For VIRapid and DIGFA, correlation between semiquantitative results and ELISA OD values were evaluated by Spearman's coefficient. Reproducibility was assessed on 16 randomly selected sera with Cohen's Kappa coefficient. Sensitivity and Specificity of VIRapid (74%, 96%) and ADAMU-CE (57%, 100%) did not differ from ELISA (69%, 96%) while DIGFA (72%, 72%) did (p = 0.045). ADAMU-CE was significantly less sensitive in the diagnosis of active cysts (p = 0.019) while DIGFA was significantly less specific (p = 0.014) compared to ELISA. All tests were poorly sensitive in diagnosing inactive cysts (33.3% ELISA and ADAMU-CE, 42.8% DIGFA, 47.6% VIRapid). The reproducibility of all RDTs was good-very good. Band intensity of VIRapid and DIGFA correlated with ELISA OD values (r = 0.76 and r = 0.79 respectively, p<0.001). CONCLUSIONS/SIGNIFICANCE: RDTs may be useful in resource-poor settings to complement ultrasound diagnosis of CE in uncertain cases. VIRapid test appears to perform best among the examined kits, but all tests are poorly sensitive in the presence of inactive cysts, which may pose problems with accurate diagnosis

Circulating small RNA profiling of patients with alveolar and cystic echinococcosis

Alveolar (AE) and cystic (CE) echinococcosis are two parasitic diseases caused by the tapeworms Echinococcus multilocularis and E. granulosus sensu lato (s. l.), respectively. Currently, AE and CE are mainly diagnosed by means of imaging techniques, serology, and clinical and epidemiological data. However, no viability markers that indicate parasite state during infection are available. Extracellular small RNAs (sRNAs) are short non-coding RNAs that can be secreted by cells through association with extracellular vesicles, proteins, or lipoproteins. Circulating sRNAs can show altered expression in pathological states; hence, they are intensively studied as biomarkers for several diseases. Here, we profiled the sRNA transcriptomes of AE and CE patients to identify novel biomarkers to aid in medical decisions when current diagnostic procedures are inconclusive. For this, endogenous and parasitic sRNAs were analyzed by sRNA sequencing in serum from disease negative, positive, and treated patients and patients harboring a non-parasitic lesion. Consequently, 20 differentially expressed sRNAs associated with AE, CE, and/or non-parasitic lesion were identified. Our results represent an in-depth characterization of the effect E. multilocularis and E. granulosus s. l. exert on the extracellular sRNA landscape in human infections and provide a set of novel candidate biomarkers for both AE and CE detection

Watch and wait approach for inactive echinococcal cyst of the liver: An update

Human cystic echinococcosis (CE) is a chronic, complex and neglected infection causing severe disease in humans. Hepatic CE cysts are detected and classified mainly by using ultrasound. Expert opinion and published data suggest that uncomplicated inactive liver cysts do not require treatment and only need to be monitored over time ("Watch and Wait"). Here we update our findings as published in 2014 on the "Watch and Wait" approach applied to inactive, asymptomatic cysts of the liver to keep the medical community informed. Clinical data of patients who accessed the World Health Organization Collaborating Center for CE at the University of Pavia-San Matteo Hospital Foundation from January 1991 to October 2017 were analyzed. Inclusion criteria were presence of one or more inactive uncomplicated cysts in the liver (CE4 or CE5), without any history of previous treatment, and an ultrasound-based follow-up of at least 24 months. Fifty-three patients with 66 inactive cysts fulfilled the inclusion criteria. Of these, 11 patients are newly described here; 37 were part of our previously described cohort and the follow-up for 17 of them was further extended; and five were excluded from the previously published analysis as their follow-up was too short, but could be included now. Without the need for treatment and without development of complications, 98.5% of cysts remained inactive over time. In only one patient (1.9% of patients), a reactivation of one cyst (1.5% of cysts) was observed

Role of microRNAs in host defense against Echinococcus granulosus infection: a preliminary assessment

Cystic echinococcosis (CE) is a neglected helminthic zoonosis caused by the larval stage of the tapeworm Echinococcus granulosus s.l. MicroRNAs (miRNAs) are regulators of gene expression that have been linked with the pathogenesis of several human diseases, but little exists in the available literature about miRNAs in CE. Here, we investigate the expression profiles of 84 microRNAs relevant to the function of lymphocytes and other immune cells during CE infection in the peripheral blood of patients with cysts in active and inactive stages. We applied the microRNA PCR array technology to blood samples from 20 patients with a single hepatic CE cyst in either the active (CE3b) or inactive (CE4–CE5) stage. Our results show a significant upregulation of eight miRNAs (let-7g-5p, let-7a-5p, miR- 26a-5p, miR- 26b-5p, miR- 195-5p, miR- 16-5p, miR- 30c-5p, and miR- 223-3p) in patients with active cysts compared to those with inactive cysts. The high expression of these miRNAs in patients with active cysts suggests their role in a specific host immune response against the infection. Further work in this direction may help shed light on the pathogenesis of human CE.Fil: Mariconti, Mara. Universita Degli Studi Di Pavia; ItaliaFil: Vola, Ambra. San Matteo Hospital Foundation; ItaliaFil: Manciulli, Tommaso. Universita Degli Studi Di Pavia; ItaliaFil: Genco, Francesca. San Matteo Hospital Foundation; ItaliaFil: Lissandrin, Raffaella. Universita Degli Studi Di Pavia; ItaliaFil: Meroni, Valeria. Universita Degli Studi Di Pavia; ItaliaFil: Rosenzvit, Mara Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; ArgentinaFil: Tamarozzi, Francesca. No especifíca;Fil: Brunetti, Enrico. Universita Degli Studi Di Pavia; Itali

Long-term Sonographic and Serological Follow-up of Inactive Echinococcal Cysts of the Liver: Hints for a "Watch-and-Wait" Approach.

none7Human cystic echinococcosis is a chronic, complex and neglected infection. Its clinical management has evolved over decades without adequate evaluation of efficacy. Recent expert opinion recommends that uncomplicated inactive cysts of the liver should be left untreated and solely monitored over time ("watch-and-wait" approach). However, clinical data supporting this approach are still scant and published mostly as conference proceedings. In this study, we report our experience with long-term sonographic and serological follow-up of inactive cysts of the liver. From March 1994 to October 2013, 38 patients with 47 liver cysts, diagnosed as inactive without any previous treatment history, were followed with ultrasound and serology at 6-12 months intervals for a period of at least 24 months (median follow-up 51.95 months) in our outpatient clinic. In 97.4\% of patients, the cysts remained inactive over time and in only one case was reactivation of the cyst detected. No complications occurred during the time of monitoring. During follow-up, serology tests for CE were negative at diagnosis or became negative in 74.1\% and were positive or became positive in 25.9\% of cases. Patients with inactive cysts on ultrasound but positive serological tests were also investigated by CT scan (chest and abdomen) to rule out extra-hepatic cyst localization. This study confirms the importance of a stage-specific approach to the management of cystic echinococcosis and supports the use of a monitoring-only approach to inactive, uncomplicated cysts of the liver. It also confirms that serology plays only an ancillary role in the clinical management of these patients, compared to ultrasound and other imaging techniques. The implications of these findings for clinical management and natural history of cystic echinococcosis are discussed.L. Piccoli;F. Tamarozzi;F. Cattaneo;M. Mariconti;C. Filice;A. Bruno;E. BrunettiL., Piccoli; F., Tamarozzi; F., Cattaneo; M., Mariconti; Filice, Carlo; A., Bruno; Brunetti, Enric