The Human Metapneumovirus Matrix Protein Stimulates the Inflammatory Immune Response In Vitro

Each year, during winter months, human Metapneumovirus (hMPV) is associated with epidemics of bronchiolitis resulting in the hospitalization of many infants. Bronchiolitis is an acute illness of the lower respiratory tract with a consequent inflammation of the bronchioles. The rapid onset of inflammation suggests the innate immune response may have a role to play in the pathogenesis of this hMPV infection. Since, the matrix protein is one of the most abundant proteins in the Paramyxoviridae family virion, we hypothesized that the inflammatory modulation observed in hMPV infected patients may be partly associated with the matrix protein (M-hMPV) response. By western blot analysis, we detected a soluble form of M-hMPV released from hMPV infected cell as well as from M-hMPV transfected HEK 293T cells suggesting that M-hMPV may be directly in contact with antigen presenting cells (APCs) during the course of infection. Moreover, flow cytometry and confocal microscopy allowed determining that M-hMPV was taken up by dendritic cells (moDCs) and macrophages inducing their activation. Furthermore, these moDCs enter into a maturation process inducing the secretion of a broad range of inflammatory cytokines when exposed to M-hMPV. Additionally, M-hMPV activated DCs were shown to stimulate IL-2 and IFN-γ production by allogeneic T lymphocytes. This M-hMPV-mediated activation and antigen presentation of APCs may in part explain the marked inflammatory immune response observed in pathology induced by hMPV in patients

Modulation of vaccine-induced immune responses to hepatitis C virus in rhesus macaques by altering priming before adenovirus boosting

BACKGROUND: Preventive and therapeutic vaccine strategies aimed at controlling hepatitis C virus (HCV) infection should mimic the immune responses observed in patients who control or clear HCV, specifically T helper (Th) type 1 and CD8+ cell responses to multiple antigens, including nonstructural protein (NS) 3. Given the experience with human immunodeficiency virus, the best candidates for this are based on DNA prime, pox, or adenovirus boost regimens. METHODS: In rhesus macaques, we compared NS3-expressing DNA prime and adenovirus boost strategy with 2 alternative priming approaches aimed at modifying Th1 and CD8+ responses: DNA adjuvanted with interleukin (IL)-2- and -12-encoding plasmids or Semliki Forest virus (SFV). RESULTS: All prime-boost regimens elicited NS3-specific B and T cell responses in rhesus macaques, including CD8+ responses. SFV priming induced higher lymphoproliferation and longer Th1 memory responses. The use of IL-2- and IL-12-expressing vectors resulted in reduced Th2 and antibody responses, which led to increased Th1 skewing but not to an increase in the magnitude of the IFN- gamma and CD8+ responses. CONCLUSIONS: All strategies induced Th1 cellular responses to HCV NS3, with fine modulations depending on the different priming approaches. When they are developed for more HCV antigens, these strategies could be beneficial in therapeutic vaccine approaches

Expanded tracking of a Beijing Mycobacterium tuberculosis strain involved in an outbreak in France

14 páginas, 3 figuras, 1 tablaAdvanced epidemiological surveillance supported on genomic epidemiology were necessary to fully describe a Mycobacterium tuberculosis Beijing strain (ARA, Auvergne-Rhône-Alpes region) outbreak in France. The index case was a migrant from Cape Verde with a two-year history of tuberculosis, high bacillary load, and frequent trips within France and to Portugal. We designed an ARA-specific PCR to complete the tracking of this outbreak strain. The ARA-specific PCR was applied on 160 Beijing isolates from independent cases from the Auvergne-Rhône-Alpes region and on 25 cases from Cape Verde migrants in the Île-de-France region. No more cases were found, indicating that the previous surveillance had been exhaustive enough to capture the whole dimension of the outbreak in France. Next, we performed a cross-border surveillance, applying the PCR on 38 Beijing isolates in Portuthe gal. In all four cases (all migrants from Cape Verde) the ARA strain was identified. Whole genome sequences analysis of all ARA isolates representatives indicated a diversification of the strain into two variants present one in France and one in PortugalThis work was supported by ERANet-LAC [ELAC2015/T08-0664] and Instituto de Salud Carlos III [AC16/00057, FIS15/01554, FIS13/ 01207, CP15/00075, PI16/01449, 1PI19/00331] and cofounded by European Regional Development Funds from the European Commission: “A way of making Europe”. Miguel Servet Contract CP15/00075 and CPII20/00001) to LPL. JP [CEECIND/00394/2017] is supported by FCT through Estímulo Individual ao Emprego Cientifico.Peer reviewe

BiCMOS Implementation of a Full-digital Linearized System for Complex Modulation Transmitter

International audienceThis paper presents an original way to implement a wide band, low power, full-digital, linearized system for complex modulation transmitter. The circuit is devoted to all applications that require efficient amplification. It has been implemented ina 0.13µm BiCMOS9 process, so simulation and experimental results are presented to validate the principle of the system

BiCMOS Implementation of a Full-digital Linearized System for Complex Modulation Transmitter

International audienceThis paper presents an original way to implement a wide band, low power, full-digital, linearized system for complex modulation transmitter. The circuit is devoted to all applications that require efficient amplification. It has been implemented ina 0.13µm BiCMOS9 process, so simulation and experimental results are presented to validate the principle of the system

Актуальність використання GenoType MTBDRplus для ранньої діагностики мультирезистентних форм туберкульозу

The type of diagnostics methods and their duration, loss of time for the appointment of the optimal treatment are the major categories of factors that influence on multidrug resistant tuberculosis treatment effectiveness. Problem of multidrug resistant tuberculosis and improving of its laboratory diagnosis is very important in Ukraine. Inadequate and ineffective treatment is applied in cases of delayed diagnostic of multidrug resistant tuberculosis in patient. It causes further spread of drug-resistant tuberculosis and increase of mycobacterium tuberculosis drug resistance to anti-tuberculosis drugs. Therefore, rapid diagnostics of multidrug resistant tuberculosis is a requirement for timely and correct treatment strategy, and one of the urgent problems of modern phthisiology.The aim of this review was to evaluate the GenoType MTBDRplus for early diagnosis of multidrug-resistant tuberculosis diagnostic in Ukraine.Conclusions. Use of the GenoType MTBDRplus (2.0) assay for rapid multidrug resistant tuberculosis detection in Ukraine is important. With Genotype MTBDRplus test TB Complex identification and resistance detection to INH and RMP is completed in 8 hours compared to 25 days in Bactec MGIT 960 System and three months on solid LG medium. Pulmonary specimens (sputum, bronchoalveolar lavage, bronchoscopic aspirate), as well as clinical specimens from extrapulmonary sites (pleural fluid, lymph node biopsy, ascitic fluid, cerebrospinal fluid) can be used. Smear-positive patients, as well smear-negative and pulmonary cases as well extra-pulmonary, would be to benefit from using the revised version 2.0 of MTBDRplus. The sensitivity of this method is 95 to 97 % and the specificity – up to 90.7 %. It is recommended for detection of MDR TB in contact persons, for screening of multi-resistant tuberculosis patients and for detection of multi-resistant tuberculosis in TB patients with treatment failure, for determination of genotype of Mycobacterium tuberculosis in the region. Qualified bacteriologic diagnostics has the potential to interrupt the transmission chain of resistant M. tuberculosis.Одними из основных факторов, которые влияют на эффективность лечения больных мультирезистентным туберкулёзом, являются длительность и вид его диагностики, потеря времени до назначения оптимального лечения для этой категории больных. В Украине проблема мультирезистентного туберкулёза с усовершенствованием его лабораторной диагностики очень актуальна. При несвоевременной диагностике мультирезистентного туберкулёза применяется неадекватное и неэффективное лечение. Это является причиной дальнейшего распространения резистентных форм туберкулёза и нарастания медикаментозной резистентности микобактерий туберкулёза к противотуберкулёзным препаратам. Поэтому экспресс-диагностика мультирезистентного туберкулёза – необходимое условие для своевременной и правильной тактики лечения и одна из актуальных проблем современной фтизиатрии.Цель работы – обзор специализированной научной литературы на предмет установления актуальности использования GenoType MTBDRplus для ранней диагностики мультирезистентных форм туберкулёза в Украине.Выводы. Использование диагностического экспресс-теста Genotype® MTBRplus (2.0) в Украине является актуальным. Этот метод позволяет сократить сроки идентификации М. tuberculosis complex и установить резистентность не только к Rif, но и к изониазиду. Это даёт возможность в течение 2 суток установить диагноз мультирезистентного туберкулёза (при классических методах – до 3 месяцев) и начать оптимальный режим противотуберкулёзной химиотерапии. Единственный из методов, при котором можно использовать и положительные, и отрицательные мазки, что позволяет использовать набор для скрининга туберкулёза. Прямым материалом являются не только мокроты, но и бронхоальвеолярный смыв, плевральная и спинномозговая жидкость от больных как лёгочными формами туберкулёза, так и внелёгочными. Диагностическая чувствительность теста составляет 95–97 %, а диагностическая специфичность – 90,7 %. Рекомендуется использовать для выявления мультирезистентного туберкулёза у контактных лиц, скрининга мультирезистентного туберкулёза у больных и выявления мультирезистентного туберкулёза у больных туберкулёзом с неудачами лечения; определения генотипических характеристик микобактерии туберкулёза в регионе. Качественная и быстрая бактериологическая диагностика туберкулёза является залогом улучшения эпидемиологической ситуации в стране.Одними з основних чинників, які впливають на ефективність лікування хворих на мультирезистентний туберкульоз, є тривалість і вид його діагностики, втрата часу до призначення оптимального лікування для цієї групи хворих. В Україні проблема мультирезистентного туберкульозу з удосконаленням його лабораторної діагностики – дуже актуальна. Внаслідок несвоєчасної діагностики мультирезистентного туберкульозу хворому застосовується неадекватне та неефективне лікування. Це є причиною дальшого поширення резистентних форм туберкульозу та зростання медикаментозної резистентності мікобактерій туберкульозу до протитуберкульозних препаратів. Тому експрес-діагностика мультирезистентного туберкульозу є необхідною умовою для своєчасної та правильної тактики лікування та однією з актуальних проблем сучасної фтизіатрії.Мета роботи – огляд спеціалізованої наукової літератури щодо встановлення актуальності використання GenoType MTBDRplus для ранньої діагностики мультирезистентних форм туберкульозу в Україні.Висновки. Використання діагностичного експрес-тесту Genotype® MTBRplus (2.0) в Україні є актуальним. Цей метод дає можливість скоротити терміни ідентифікації М. tuberculosis complex і встановлювати резистентність не тільки до Rif, а й до ізоніазиду. Отже, можна протягом 2 діб встановити діагноз мультирезистентного туберкульозу (при класичних методах – до 3 місяців) і розпочати оптимальний режим протитуберкульозної хіміотерапії. Це – єдиний із методів, при котрому можна використовувати як позитивні, так і негативні мазки, що дає змогу застосовувати набір для скринінгу туберкульозу. Прямим матеріалом є не тільки харкотиння, а й бронхоальвеолярний змив, плевральна та спинномозкова рідина від хворих як легеневими формами туберкульозу, так і позалегеневими. Діагностична чутливість тесту становить 95–97 %, а діагностична специфічність – 90,7 %. Рекомендовано використовувати для виявлення мультирезистентного туберкульозу в контактних осіб, скринінгу мультирезистентного туберкульозу у хворих і виявлення мультирезистентного туберкульозу у хворих на туберкульоз із невдачами лікування; визначення генотипових характеристик мікобактерії туберкульозу у регіоні. Якісна та швидка бактеріологічна діагностика туберкульозу  – запорука поліпшення епідеміологічної ситуації у країні

High-throughput mycobacterial interspersed repetitive-unit-variable-number tandem-repeat genotyping for Mycobacterium tuberculosis epidemiological studies

International audienceThe emergence of drug-resistant forms of tuberculosis (TB) represents a major public health concern. Understanding the transmission routes of the disease is a key factor for its control and for the implementation of efficient interventions. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) marker typing is a well-described method for lineage identification and transmission tracking. However, the conventional manual genotyping technique is cumbersome and time-consuming and entails many risks for errors, thus hindering its implementation and dissemination. We describe here a new approach using the QIAxcel system, an automated high-throughput capillary electrophoresis system that also carries out allele calling. This automated method was assessed on 1,824 amplicons from 82 TB isolates and tested with sets of markers of 15 or 24 loci. Overall allele-calling concordance between the methods from 140 to 1,317 bp was 98.9%. DNA concentrations and repeatability and reproducibility performances showed no biases in allele calling. Furthermore, turnaround time using this automated system was reduced by 81% compared to the conventional manual agarose gel method. In sum, this new automated method facilitates MIRU-VNTR genotyping and provides reliable results. Therefore, it is well suited for field genotyping. The implementation of this method will help to achieve accurate and cost-effective epidemiological studies, especially in countries with a high prevalence of TB, where the high number of strains complicates the surveillance of circulating lineages and requires efficient interventions to be carried out in an urgent manner

The effectiveness of GenoType MTBDRplus using in the diagnosis of tuberculosis in Zaporizhzhia region

The method GenoType MTBDRplus, v.2 allows to study clinical material from positive and negative smears, to examine patients with new and previously treated cases of tuberculosis pulmonary and extrapulmonary localization. But now there is a small amount of researches concerning the use of this method in the diagnosis of tuberculosis. Aim. To evaluate the effectiveness of GenoType MTBDRplus, v.2 method using in clinical material research due to the diagnosis of tuberculosis in the Zaporizhzhia region in comparison with standard methods of investigation. Materials and methods. The analysis of 52 results of studies of clinical material using the GenoType MTBDRplus test system, v.2 from patients who were examined and treated in dispensaries of the Zaporizhzhia region in 2016 was carried out. There were 67.3 % of men and 32.7 % of women among the patients. The mean age of the patients was 46.0 ± 1.9 years. The study of the clinical material using the GenoType MTBDRplus test, v.2 was performed according to the standard instructions. Results. The results were positive in 12.5 % of the cases when tested by GenoType MTBDRplus, v.2 (p ˂ 0.05) among negative results of the microscopy of the clinical material. The proportion of coincidence between the cultural and molecular genetic methods was 90 % for sputum and 100 % for other clinical material. In the presence of multiresistance, both methods of investigation coincided in the number of detected cases (50 % for the GenoType MTBDRplus method, v.2 and 42.9 % for the cultural method, respectively, p ˂ 0.05). In comparison with the results of the cultural method, the GenoType MTBDRplus test, v.2 made it possible to establish resistance to rifampicin in 35.7 % (p < 0.05) of patients whose resistance was not determined. False-positive and false-negative results are due, perhaps, to the heterogeneity of strains of mycobacterium tuberculosis in clinical specimens, the absence of viable mycobacteria, the absence of phenotypic manifestations of mycobacterium tuberculosis genetic mutations, and errors in the investigations. The sensitivity of the GenoType MTBDRplus test, v.2 is about 78 %, but the specificity reaches 97 %. At the same time, the sensitivity of the test for multidrug resistance determining reaches 100 %, the specificity is about 89 %. The greater positive predictive value for cases in which the DNA of mycobacteria is determined and the negative predictive value is for cases in which multidrug resistance is determined. Conclusions. This indicates that the using of the GenoType MTBDRplus test, v.2, together with the cultural method of investigation, has a high diagnostic value, high sensitivity (100 %) and specificity (89 %) for the definition of multiresistance, and makes it possible to obtain a result in a short time compared to culture. tuberculosis; diagnostic techniques and procedures; genotype; efficienc

False-Positive Results in a Recombinant Severe Acute Respiratory Syndrome-Associated Coronavirus (SARS-CoV) Nucleocapsid-Based Western Blot Assay Were Rectified by the Use of Two Subunits (S1 and S2) of Spike for Detection of Antibody to SARS-CoV

To evaluate the reactivity of the recombinant proteins expressed in Escherichia coli strain BL21(DE3), a Western blot assay was performed by using a panel of 78 serum samples obtained, respectively, from convalescent-phase patients infected with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (30 samples) and from healthy donors (48 samples). As antigen for detection of SARS-CoV, the nucleocapsid protein (N) showed high sensitivity and strong reactivity with all samples from SARS-CoV patients and cross-reacted with all serum samples from healthy subjects, with either those obtained from China (10 samples) or those obtained from France (38 serum samples), giving then a significant rate of false positives. Specifically, our data indicated that the two subunits, S1 (residues 14 to 760) and S2 (residues 761 to 1190), resulted from the divided spike reacted with all samples from SARS-CoV patients and without any cross-reactivity with any of the healthy serum samples. Consequently, these data revealed the nonspecific nature of N protein in serodiagnosis of SARS-CoV compared with the S1 and S2, where the specificity is of 100%. Moreover, the reported results indicated that the use of one single protein as a detection antigen of SARS-CoV infection may lead to false-positive diagnosis. These may be rectified by using more than one protein for the serodiagnosis of SARS-CoV