Real-time PCR Kit Research Articles

Surveillance of the equine infectious anemia virus in Eastern and Central Saudi Arabia during 2014-2016.

Background:Equine infectious anemia virus (EIAV) is one of the most important threats to the equine industry globally. This is due to the poor performance of the affected horses, which requires euthanization of the infected animals upon the infection confirmation. Infected animals remain carriers throughout their life. EIAV infection has been reported in many parts of the world, including North America, Europe, Asia, and Africa. However, the EIAV status is never assessed in horses in the Gulf area, especially in the Kingdom of Saudi Arabia (KSA).Aim:This study aimed to perform molecular and serological surveillance among some horse populations in Eastern and Central Saudi Arabia.Materials and Methods:Sera and whole blood were collected from 361 horses and 19 donkeys from the eastern and central regions of Saudi Arabia during January 2014-December 2016. Sera were tested by the commercial EIAV enzyme-linked immunosorbent assay kits. Moreover, the collected blood samples were tested by the commercial real-time polymerase chain reaction kits.Results:Our serological surveillance revealed the absence of any antibodies against EIAV in the tested animals. Similar results were reported for the tested horses’ plasma. This study confirms the absence of EIAV in horses and donkeys from Eastern and Central Saudi Arabia during the tenure of the current study. However, careful monitoring of the EIAV is highly recommended to avoid the emergence of such a virus in the horse population in Saudi Arabia.Conclusion:To the best of our knowledge, this is the first EIAV surveillance conducted not only in Saudi Arabia but also in the Gulf area. This study confirms the absence of EIAV in the tested equine population in the eastern and central regions of Saudi Arabia during 2014-2016.

Assessment of the first commercial multiplex PCR kit (ParaGENIE Crypto-Micro Real-Time PCR) for the detection of Cryptosporidium spp., Enterocytozoon bieneusi, and Encephalitozoon intestinalis from fecal samples

Cryptosporidium spp. Enterocytozoon bieneusi and Encephalitozoon intestinalis are opportunistic pathogens responsible for gastrointestinal diseases. We evaluated the ParaGENIE Crypto-Micro Real-Time PCR kit (Ademtech, France), the first CE-IVD compliant PCR assay available for these pathogens. This study was conducted blindly against a reference panel of 115 stool specimens including positive samples for Cryptosporidium spp. (n = 48) and E. bieneusi (n = 38) as well as negative or positive samples for other parasites to test for cross-reactivity. An additional set of samples corresponding to 8 rare Cryptosporidium species was also included. Discrepancies were evaluated with external in-house PCR tests. The ParaGENIE Crypto-Micro PCR assay displayed a sensitivity/specificity of 91.7%/100% and 97.3%/98.7% for Cryptosporidium spp. and E. bieneusi, respectively, and was able to detect all 12 Cryptosporidium species of the reference panel, including rare species. This new CE-IVD assay will facilitate the diagnosis of intestinal cryptosporidiosis and microsporidiosis, a major concern in immunocompromised patients and travelers.

Screening methods to identify the complex of strains of Plum pox virus (PPV)

From the 10 known strains of thePlumpox virus (PPV) in the Russian Federation 7 strains are spread – D, M. Rec, W, C, CV, and CR with the prevalence of D and W strains. A characteristic feature of the Plum pox virus population in the Russian Federation is the wide spread of W, C and CR strains, extremely rare or not found at all in other regions of the world. Screening tests for detection of the complex of PPV strains by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) were carried out. It has been established that effective detection of PPV-D, PPV-M, PPV-Rec, PPV-C, PPV-CR, PPV-EA, PPV-W and PPV-T strains is achieved using PPV-ELISA test systems with polyclonal antibodies from ACD, Adgen, Agdia, Bioreba, DSMZ and Loewe companies. Monoclonal antibodies 5B-IVIA do not allow to diagnose isolates related to the strain PPV-CR. For universal detection of isolates of all PPV strains, it is necessary to use a real-time PCR kit for PPV (Syntol), a FLASH-PCR kit for PPV (Agrodiagnostica) or conventional PCR with one of the following primer pairs: P1/P2, s1/as2 or 3´NCP sense/3´NCP antisense.

Shedding and genetic diversity of Coxiella burnetii in Polish dairy cattle.

Q fever is a worldwide zoonotic disease reported in humans and many animal species including cattle. The aims of this study were to evaluate the prevalence of Coxiella (C.) burnetii shedding in Polish dairy cattle herds and to identify the pathogen’s genotypes and sequence types (STs) using multiple-locus variable number tandem repeat analysis (MLVA) and multispacer sequence typing (MST) methods. The presence of C. burnetii DNA was detected using a commercial real-time PCR kit, targeting the IS1111 element. Overall, 1,439 samples from 279 herds were tested including: 897 individual milk specimens, 101 bulk tank milk samples, 409 genital tract swabs and 32 placentas. Furthermore, 30 consumer milk samples, including 10 from vending machines and 77 dairy products were also analyzed. C. burnetii shedding was confirmed in 31.54% of tested cattle herds as well as in 69.16% of consumer milk and dairy products. Among real-time PCR–positive samples, 49 specimens obtained from 49 cattle herds and 8 samples of purchased dairy products were selected for genotyping. Overall, five previously known MLVA genotypes (I, J, BG, BE, and NM) and three new ones (proposed as PL1, PL2, and PL3) were identified. Two MST sequence types were recorded: ST16 and a novel sequence (ST61). The new genotypes and sequence types need further research particularly into their pathogenicity to humans.

Viral and Atypical Bacterial Respiratory Infections in a University Teaching Hospital.

Respiratory viral and atypical bacterial agents lead to infections in a large spectrum, from mild symptoms to respiratory failure. In the present study, we aimed to detect multiple viral and bacterial agents in the respiratory samples of inpatients by real-time polymerase chain reaction (RT-PCR). Nasopharyngeal swabs and broncho-alveolar lavage samples from inpatients with respiratory infection symptoms at the Uludag University Hospital between December 1, 2015 and March 31,2018 were investigated. DNA/RNA was extracted using the EZ1 Virus Mini Kit v2.0 (Qiagen, Belgium) with the EZ1 extraction device (Qiagen, Belgium). The R-GENE® RT-PCR (Biomerioux, France) kit was used to detect influenza A, influenza B, respiratory syncytial virus (RSV), human metapneumovirus, rhinovirus/enterovirus (RV/EV), adenovirus, human bocavirus (hBoV), corona virus, parainfluenza virus, Chlamydia pneumoniae/Mycoplasma pneumoniae, and Legionella pneumophila in Rotor-Gene Q (Qiagen, Belgium). Patients were aged between 0 and 90 years. Overall, 177 (56.9%) patients were men and 134 (43.1%) were women. A total of 311 samples were analyzed, of which 214 (68.8%) were positive. In total, 360 agents, including 338 viruses and 22 bacteria, were detected. The commonest agents were influenza A+B (n = 65, 18,1%), hBoV (n = 64, 17.8%), RV/EV (n = 56, 15.6%), and RSV (n = 47, 13.1%). Rapid diagnosis of viral infections by RT-PCR is important for the specific treatment of patients.

The qPCR analysis of vaginal microflora for the diagnosis of bacterial vaginosis

The correct information about of the vaginal microflora plays an important role in preventing the occurrence of urinary tract infections and sexually transmitted infections among women. Disbalance of obligate and facultative microflora causes disbacteriosis, a risk factor for emergence of infectious diseases. It is known that the cause of bacterial vaginosis (BV) is not a single pathogen but a impairments in of the general balance of the vaginal microflora, which manifests a decrease of the normal microflora (Lactobacillus spp) and intense increase of pathogenic aerobic and anaerobic bacteria. The development of molecular genetic analysis methods, in particular, approaches based on the use of polymerase chain reaction (PCR), significantly expanded understanding of the diversity of microbial biotopes, including identification of the key and new «players» in the development of BV. The aim of our study was to evaluate the performance of real-time PCR kit «Femoscreen» («Lytech», Russia) for comprehensive BV diagnosis.

Caspase 3/ROCK1 pathway mediates high glucose-induced platelet microparticles shedding

BackgroundPlatelet microparticles (PMPs) are closely associated with diabetic macrovascular complications. This study aimed to explore the underlying mechanisms of high glucose-induced PMPs generation. MethodsWashed platelets, obtained from the plasma of healthy male Sprague-Dawley rats, were incubated with high glucose. PMPs were isolated using gradient centrifugation and counted by flow cytometry. Expression and activity of ROCK1 and caspase3 were evaluated by real-time PCR, Western blotting, and activity assay kit. ResultsHigh glucose enhanced PMPs shedding in the presence of collagen. The mRNA and protein levels of ROCK1, but not ROCK2, were increased in platelets incubated with high glucose. Y-27632, an inhibitor of ROCK, blocked the increased PMPs shedding induced by high glucose. Expression and activity of caspase3 were elevated in platelets under the high glucose conditions. Z-DVED-FMK, a caspase3 inhibitor, inhibited ROCK1 activity and decreased the PMPs generation under high glucose. ConclusionHigh glucose increased PMPs shedding via caspase3-ROCK1 signal pathway.

Evaluation of PCR-based methods for the identification of enteroaggregative hemorrhagic Escherichia coli in sprouts

In this study real-time PCR assays were evaluated for the detection of enteroaggregative hemorrhagic Escherichia coli (EAHEC) O104:H4 in artificially contaminated mung bean and/alfalfa sprouts inoculated with 1, 10, and 100 CFU of EAHEC O104:H4 per 25 g sample (20, 10, and 2 replicates respectively). After selective culture enrichment the samples were tested using commercial real-time PCR kits detecting aggR/aaiC, stx/eae, and wzxO104. Using the commercial real-time PCR kits, the artificially contaminated samples were detected in the range of 75–80% positive results when contaminated with approximately 1 CFU, and 100% at 10 and 100 CFU. Microbiological detection employing O104-specific immunomagnetic capture and plating onto chromogenic media (modified Rainbow Agar and CHROMagar STEC) and confirmation by latex agglutination and PCR gave similar results (Cohen's kappa value between 0.61 and 1). In addition, the real-time PCR assay targeting the aggR and aaiC genes, indicative of enteroaggregative Escherichia coli (EAggEC), was tested against a panel of 60 bacterial strains and demonstrated 100% exclusivity (54 strains) and 100% inclusivity (6 strains). This study demonstrates the efficacy of the real-time PCR assays for the specific and sensitive detection of EAHEC from spouts.

P138 Clinical characteristics of patients who manifest unexplained crossmatch results for solid organ transplantation

Aim The flow cytometric crossmatch (FCXM) and the virtual crossmatch (VCXM) assess compatibility between solid organ donors and recipients. Theoretically, both methods should give the same results. Realistically, however, they can be discrepant due to limitations of assays used to detect alloantibodies, non-HLA alloantibodies, autoantibodies, and non-specific reactivity. We sought to identify clinical features of patients who manifest such discrepancies, hypothesizing an association with female gender, autoimmune disease, and potential heart recipients. Methods We retrospectively evaluated all living and deceased donor FCXMs (performed using BD FacsCanto flow cytometer, BD Biosciences, San Jose, CA) for solid organ transplant offers for patients at University of California San Diego from 2015 to 2017. Based on previous validation studies, a positive FCXM was coded as ”expected” when a single donor-specific HLA antibody (detected using LABScreen single antigen assay, One Lambda, Canoga Park, CA) demonstrated a mean fluorescent intensity of 3,000 or higher, or when the sum of multiple antibodies reached that threshold. Deceased donor typing was performed using LinkSeq real-time polymerase chain reaction kit (Linkage Biosciences, South San Francisco, CA). Living donor HLA genotyping was performed by SSO or NGS-based methods. FCXMs with unexpected positive results were investigated. Results 1594 FCXMs (1369 for kidney, 139 for heart, 47 for lung, and 39 for multiple organs) for 967 potential recipients (621 males, 346 females) were analyzed. Unexpected positive results occurred in 109 T cell FCXMs in 81 patients (8%) and 40 B cell FCXMs in 23 patients (2%). Analysis by both crossmatch and patient demonstrated correlations with female gender (T cell p Conclusions Unexpected positive FCXMs correlate with female gender, but not with transplant organ. In 5 cases with persistent unexpected positive FCXMs, we have successfully transplanted patients using the VCXM.

INVOLVEMENT OF JNK MAPK CASCADES IN THE FORMATION OF ADHESIONS IN THE ABDOMINAL CAVITY

Adhesive process in the abdominal cavity is one of the most frequent complications of intra-abdominal surgery.The aim of the study was to evaluate the activation of the JNK MAPK cascade during the experimental adhesion process.Materials and methods. Experimental studies were performed on 40 male Wistar rats (9 months old). We modeled the adhesive process in the abdominal cavity. At the time from 2 hours (2, 6, 12 hours) to 30 days (1, 3, 7, 14, 30 days), a JNK MAPK cascade was evaluated in the zone of damage to the serosa using immunohistochemical methods (painting on JNK1 Phospho (pT183) / JNK2 Phospho (pT183) / JNK3 Phospho (pT221)) and real-time PCR (the MAP Kinase Signaling Pathway RT2 - Profiler ™ PCR Array (Qiagen) kit). Results. We have found that the expression of the phosphorylated part of the JNK MAPK cascades has an undulating dynamics with two peaks on the 3rd and 14th day. It has been determined that all three JNK cascades are involved in the process, and all JNK cascades are synchronously activated on the 3rd day (JNK1 increases 12-fold in comparison with the group of intact animals, JNK2 is 8-fold, JNK3 is 10-fold). On the 14th day, the activity of the JNK3 cascade is the most intensively increased – 30 times (for JNK1 – about 6 times, JNK2 – 12 times). Conclusion. Significant activation of MARK cascades has been established on the 14th day after traumatic injury, which indicates the need for long-term prevention of posttraumatic changes, in particular, the formation of adhesions.

Vérification en portée A d’une méthode de PCR triplexe pour la détection de Chlamydia trachomatis, Neisseria gonorrhoeae et Mycoplasma genitalium

Method verification, in range A, of a triplex real time PCR for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitaliumIn 2017, at the Beauvais hospital laboratory, a real time PCR system has been acquired. This work describes the method verification of a real time PCR kit, Urethritis basic®, FTD, in range A, for the qualitative detection of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Mycoplasma genitalium (MG). According to the ISO 15189 standard, a risk analysis and a verification of some performance criteria are performed on site. Intermediate precision for NG is evaluated on two samples giving equivocal results. Following runs gives negative results. Comparison between the new and the previous end-point PCR method (GenoQuick®CT, Hain Lifescience) for the detection of CT is performed on 46 samples. Four samples show discordant results (all positive with the previous method and negative with the new one). Accuracy is assessed by two EQA (External Quality Assessment) for CT and NG and gave expected results. Our tests of carry-over contamination, by alternating positive and negative samples, did not identify any contamination. Finally, we implemented continuous verification by Levey-Jennings charts established using threshold cycles of controls. This work let to conclude on the ability of the method while confirming the interest of controlling equivocal NG results. In addition, it provides a tool to continuously monitor the performance.

Evaluation of Two Commercial Real-Time PCR Kits for Aspergillus DNA Detection in Bronchoalveolar Lavage Fluid in Patients with Invasive Pulmonary Aspergillosis

Invasive pulmonary aspergillosis (IPA) is a common complication of immunosuppression. Rapid diagnosis using molecular techniques is essential to improve patient survival. PCR techniques are promising in enhancing Aspergillus detection in blood and respiratory samples. We evaluate for the first time the performances of two commercial real-time PCR kits, the A. fumigatus Bio-Evolution and the MycoGENIE A. fumigatus for the detection of A. fumigatus DNA in bronchoalveolar lavage (BAL) from patients with and without IPA. Seventy-three BAL samples were included. Thirty-one of them corresponded to patients with probable IPA, 11 to patients with possible IPA, and 31 to patients without aspergillosis, according to the 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. In the probable IPA group, A. fumigatus Bio-Evolution and the MycoGENIE A. fumigatus real-time PCR kits showed a specificity of 100% and a sensitivity of 81% and 71%, respectively. The A. fumigatus Bio-Evolution detected Aspergillus DNA in the 14 BAL samples with a positive Aspergillus culture result, whereas the MycoGENIE A. fumigatus PCR result was positive only for 12. In the possible IPA group, there were no positive real-time PCR or positive Aspergillus culture results. For the patients without aspergillosis, no positive result was observed for real-time PCR kit, despite the presence of various other non-Aspergillus pathogens in this group. Our study demonstrates an excellent specificity and a good sensitivity of A. fumigatus DNA detection in BAL samples with both kits.

Evaluation of a new multiplex PCR assay (ParaGENIE G-Amoeba Real-Time PCR kit) targeting Giardia intestinalis, Entamoeba histolytica and Entamoeba dispar/Entamoeba moshkovskii from stool specimens: evidence for the limited performances of microscopy-based approach for amoeba species identification

ObjectivesBesides the potential to identify a wide variety of gastrointestinal parasites, microscopy remains the reference standard in clinical microbiology for amoeba species identification and, especially when coupled with adhesin detection, to discriminate the pathogenic Entamoeba histolytica from its sister but non-pathogenic species Entamoeba dispar/Entamoeba moshkovskii. However, this approach is time-consuming, requires a high-level of expertise that can be jeopardized considering the low prevalence of gastrointestinal parasites in non-endemic countries. Here, we evaluated the CE-IVD-marked multiplex PCR (ParaGENIE G-Amoeba, Ademtech) targeting E. histolytica and E. dispar/E. moshkovskii and Giardia intestinalis. MethodsThis evaluation was performed blindly on a reference panel of 172 clinical stool samples collected prospectively from 12 laboratories and analysed using a standardized protocol relying on microscopy (and adhesin detection by ELISA for the detection of E. histolytica) including G. intestinalis (n = 37), various amoeba species (n = 55) including E. dispar (n = 15), E. histolytica (n = 5), as well as 17 other gastrointestinal parasites (n = 80), and negative samples (n = 37). ResultsThis new multiplex PCR assay offers fast and reliable results with appropriate sensitivity and specificity for the detection of G. intestinalis and E. dispar/E. moshkovskii from stools (89.7%/96.9% and 95%/100%, respectively). Detection rate and specificity were greatly improved by the PCR assay, highlighting several samples misidentified by microscopy, including false-negative and false-positive results for both E. dispar/E. moshkovskii and E. histolytica. ConclusionGiven the clinical relevance of amoeba species identification, microbiologists should be aware of the limitations of using an algorithm relying on microscopy coupled with adhesin detection by ELISA.

Identification and quantification of Chimerism in bone marrow transplants leukemic patients

Quantification of chimerism after bone marrow transplantation is essential for evaluation the successful of engraftment after allogenic stem cells transplantation. The aim of this work is to use STR typing test for identification and quantification the chimerism in bone marrow transplant patients. Two patients subjected to bone marrow transplantation were analyzed. DNA extracted from patients (recipient) blood, cheek swabs and donor cheek swabs. Quantifiler Real time PCR kit was used for DNA quantification. Powerplex21 kit was used for amplification STR loci. STR profiling was performed by Genetic analyzer. DNA extraction and quantification were successfully performed with suitable quantity and purity. STR loci fully amplified and analyzed. In both cases recipient and Donor shared alleles with no DNA mixtures. Few loci showed Type 3 chimerism. In conclusion the results showed that the test successfully identify used for identification and quantification of chimerism in recipient patients. List of abbreviation: DNA= Deoxyribonucleic acid, STR- short tandem repeat, SCT= stem cells transplantation.

Evaluation of EZplex MTBC/NTM Real-Time PCR kit: diagnostic accuracy and efficacy in vaccination.

PurposeTuberculosis (TB) is mainly caused by Mycobacterium tuberculosis, which is a pathogenic mycobacterial species grouped under Mycobacterium tuberculosis complex (MTBC) with four other pathogenic mycobacterial species. The mycobacteria not included in MTBC are known as nontuberculous mycobacteria (NTM), and cause several pulmonary diseases including pneumonia. Currently, NTM occurrences in TB-suspected respiratory specimens have increased, due to which, precise detection of MTBC and NTM is considered critical for the diagnosis and vaccination of TB. Among the various methods available, real-time PCR is frequently adopted for MTBC/NTM detection due to its rapidness, accuracy, and ease of handling. In this study, we evaluated a new real-time PCR kit for analytical and clinical performance on sputum, bronchial washing, and culture specimens.Materials and MethodsFor assessing its analytical performance, limit of detection (LOD), reactivity, and repeatability test were performed using DNA samples. To evaluate clinical performance, 612 samples were collected and clinically tested at a tertiary hospital.ResultsLOD was confirmed as 0.584 copies/µL for MTBC and 47.836 copies/µL for NTM by probit analysis (95% positive). For the reactivity test, all intended strains were detected and, in the repeatability test, stable and steady results were confirmed with coefficient of variation ranging from 0.36 to 1.59. For the clinical test, sensitivity and specificity were 98.6%–100% and 98.8%–100% for MTBC and NTM, respectively.ConclusionThe results proved the usefulness of the kit in TB diagnosis. Furthermore, it could be adopted for the assessment of vaccine efficacy.

Modified use of real-time PCR detection of group B Streptococcus in pregnancy.

The CDC recommends antenatal screening of vaginal/rectal samples for Streptococcus agalactiae at 35-37 weeks' gestation, with intra-partum antibiotic prophylaxis for positive cases. We tested a modified use of the Cepheid Xpert GBS real-time PCR kit on enrichment cultures from 554 vaginal/rectal swabs compared to the current subculturing gold standard method. Swabs were inoculated on polymyxin nalidixic acid agar plates, and Todd-Hewitt enrichment broth cultures were examined daily for growth. Todd-Hewitt broth culture was also used for Xpert GBS. There was 92.06 % agreement between the subculture and PCR methods. Sensitivity of Xpert GBS was 100 %, specificity was 89.40 %, positive predictive value was 75.96 % and negative predictive value was 100 %. Colonization incidence was higher with younger (≤24 years) or older (≥35 years) maternal age. Modified use of the Cepheid Xpert GBS would assist rapid diagnosis of S. agalactiae colonization and facilitate timely and appropriate assignment to intra-partum antibiotic prophylaxis.

EGFR and KRAS Gene Mutations in 754 Patients with Resectable Stage I-IIIa Non-small Cell Lung Cancer and Its Clinical Significance

背景与目的表皮生长因子受体（epidermal growth factor receptor, EGFR）和KRAS基因是非小细胞肺癌（non-small cell lung cancer, NSCLC）重要的分子靶点，但目前研究主要集中在晚期NSCLC组织和血浆标本的EGFR检测，早期NSCLC组织样本中EGFR和KRAS突变特征尚不清楚。本研究将探讨Ⅰ期-Ⅲa期NSCLC EGFR和KRAS基因突变与相关临床病理特征的关系。方法采用突变扩增系统（amplification refractory mutation system, ARMS）PCR方法检测北京协和医院病理科提供的754例Ⅰ期-Ⅲa期NSCLC组织样本的EGFR和KRAS基因突变状况，分析基因突变率及其与临床病理特征的关系。结果EGFR和KRAS基因热点突变的突变率分别为34.5%和13.1%，其中有3例样本具有EGFR和KRAS基因的双突变。EGFR基因在女性中的突变率高于男性（39.5% vs 29.4%, P=0.076），在腺癌中的突变率（38.7%）高于鳞癌、腺鳞癌、大细胞癌（P < 0.01），但仍明显低于其他研究报道的亚裔晚期腺癌突变率（-50%）。KRAS基因突变在男性中的突变率高于女性（16.6% vs 9%, P=0.048），且在腺癌中的突变率也高于其他类型，但差异不显著（P=0.268）。与KRAS基因突变阳性组相比，EGFR基因突变阳性组在年龄分布上有年轻化的趋势（P=0.031, 5），在性别分布上有显著性差异（P < 0.01）。结论Ⅰ期-Ⅲa期NSCLC EGFR基因突变率较晚期患者低，且EGFR和KRAS基因双突变的发生率为0.9%。

MOLECULAR TYPES IN THE PROGNOSIS OF PAPILLARY THYROID CANCER BASED ON THE ANALYSIS OF THE STATUS OF BRAF V600E, INDEX AND KI-67 EXPRESSION

The aim of our research was to determine the possibilities to stratify patients with papillary thyroid cancer (PTC) on the group’s forecast, depending on the status of the mutation BRAF v600e and Ki-67 expression. The study included 89 PTC patients with the known prognosis for the period of 2005-2015. They were treated in the Altai regional oncology center and underwent surgery for PTC over the period of 2005-2013. Mutation p. Val600Glu (V600E) BRAF gene was determined with the use of allele-specific real-time PCR kit «Real-time-PCR-BRAF-V600E («BioLink»)». Immunohistochemical examination was performed with antibodies Ki-67 (clone MIB1, «DAKO») on stainer «Ventana XT» according to standard protocols. We revealed BRAF V600E not to be an independent predictor. Different variants of PTC, depending on the level of expression of Ki-67 in combination with a mutation of BRAF V600E was allowed to identify 3 groups of molecular types of PTC: a lack of mutation and a low Ki-67 (type I), with mutation and a low Ki-67 (type II) and with the presence and/or absence of a mutation and high level Ki-67 (type III). I molecular type PTC is the most favorable from the point of view of survival: 100% of patients live 10 years. Molecular type II is characterized by an intermediate prognosis: 76% of patients lived out up to 10 years. Molecular type III is characterized by the poor prognosis: only 36.8 percent of cases lived up to 10 years. In multivariate analysis of survival in PTC patients the independent prognostic factor happened to be the molecular type of tumor. In this case χ2 = 35 at p ≤ 0.000005.

Clinical evaluation of a new single-tube multiplex reverse transcription PCR assay for simultaneous detection of 11 respiratory viruses, Mycoplasma pneumoniae and Chlamydia in hospitalized children with acute respiratory infections

Respiratory Pathogen 13 Detection Kit (13× kit) is able to simultaneously detect 11 respiratory viruses, Mycoplasma pneumoniae (MP) and Chlamydia in a single reaction. Using 572 Nasopharyngeal aspirates collected from hospitalized children, the clinical performance of 13× kit for detecting 11 respiratory viruses was evaluated in comparison with a routinely used 2-tube multiplex reverse transcription PCR assay (2-tube assay) at provincial Centers for Disease Control and Prevention in China. The clinical performance of 13× kit for detecting MP and Chlamydia was evaluated by commercial real-time quantitative PCR (qPCR) kits or sequencing. For tested viruses, the assay concordance was 95.98% and the kappa coefficient was 0.89. All the MP and Chlamydia positive samples detected by 13× kit were confirmed as true positives. The utilization of the 13× kit in clinical settings will be helpful for doctors to assess clinical outcome according to virus type or multiple infections, and to limit the use of antibiotics.

Advanced Glycation End Product 3 (AGE3) Increases Apoptosis and the Expression of Sclerostin by Stimulating TGF-β Expression and Secretion in Osteocyte-Like MLO-Y4-A2 Cells.

Advanced glycation end products (AGEs) cause bone fragility due to deterioration in bone quality. We previously reported that AGE3 induced apoptosis and inhibited differentiation via increased transforming growth factor (TGF)-β signaling in osteoblastic cells. Additionally, we demonstrated that AGE3 increased apoptosis and sclerostin expression and decreased receptor activator of nuclear factor-κB ligand (RANKL) expression in osteocyte-like cells. However, it remains unclear whether TGF-β signaling is involved in the effects of AGEs on apoptosis and the expression of sclerostin and RANKL in osteocytes. Effects of AGE3 on apoptosis of mouse osteocyte-like MLO-Y4-A2 cells were examined by DNA fragmentation ELISA. Expression of TGF-β, sclerostin, and RANKL was evaluated using real-time PCR, Western blotting, and ELISA kits. To block TGF-β signaling, we used SD208, a TGF-β type I receptor kinase inhibitor. AGE3 (200µg/mL) significantly increased apoptosis and mRNA expression of Sost, the gene encoding sclerostin, and decreased Rankl mRNA expression in MLO-Y4-A2 cells. AGE3 significantly increased the expression of TGF-β. Co-incubation of SD208 with AGE3 significantly rescued AGE3-induced apoptosis in a dose-dependent manner. Moreover, SD208 restored AGE3-increased mRNA and protein expression of sclerostin. In contrast, SD208 did not affect AGE3-decreased mRNA and protein expression of RANKL. These findings suggest that AGE3 increases apoptosis and sclerostin expression through increasing TGF-β expression in osteocytes, and that AGE3 decreases RANKL expression independent of TGF-β signaling.