Nilo Ikuta

Evaluation and Comparison of Various PCR Methods for Detection of Mycoplasma gallisepticum Infection in Chickens

Abstract Four generic Mycoplasma gallisepticum (MG) polymerase chain reactions (PCRs) (16S rRNA PCR, three newly developed PCR methods that target surface protein genes [mgc2, LP (nested) and gapA (nested)]) were compared for analytical specificity and sensitivity and for diagnostic sensitivity (Se) and specificity of detection from tracheal swabs. The licensed MG DNA Test Kit Flock Chek test (IDEXX, Laboratories, Inc., Westbrook, ME) was as well evaluated for the diagnostic specificity and sensitivity of detection from tracheal swabs. Analytical specificity was evaluated for the four generic PCR methods using a panel of DNA samples from microorganisms that may be isolated from the trachea of commercial poultry and other fowl. PCR methods mgc2, nLP, and ngapA only amplified DNA from MG, whereas 16S rRNA PCR amplified DNA from MG and Mycoplasma imitans. The analytical sensitivity of the four generic PCR methods expressed in color-changing units (CCU)/amplification reaction was estimated for each PCR method and ranged from 4 to 400 CCU/reaction; the sensitivities of single PCR methods 16S rRNA and mgc2 were estimated at 40 CCU/reaction, the nLP at 400 CCU/reaction, and the ngapA at 4 CCU/reaction. The diagnostic sensitivity and specificity of MG detection from tracheal swab pools, as compared to isolation from choanal cleft swabs, was evaluated for the five PCR methods using three groups of birds exposed to vaccine strains ts-11 and 6/85 and to challenge strain R. All PCR methods were able to detect the vaccine strains and the challenge strain R directly from tracheal swabs, indicating that PCR primers from the different methods amplified divergent MG strains. Isolation and PCR results correlated satisfactorily among the three experimentally infected groups, with agreement values (k) ranging from 0.52 to 1.00. The ngapA, IDEXX, and mgc2 PCRs showed the best sensitivity (Se) ratios for detection of M. gallisepticum strains as compared to isolation. Compared to the ngapA and IDEXX PCR methods, the mgc2 PCR has a faster turnaround time, since this test consists of a single amplification reaction and the amplification product is detected by gel electrophoresis. Therefore, among the PCR methods evaluated in this study, the mgc2 PCR is the method of choice to further validate in the field.

Increased levels of interleukin-6, -8 and -10 are associated with fatal outcome following severe traumatic brain injury.

Abstract Background: Despite the involvement of cytokine production in neurotrauma, there is still controversy regarding cytokines levels and clinical outcome following severe traumatic brain injury (TBI). Objective: The present study was designed to investigate whether cytokine levels (of IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNF-α) are associated with primary outcome (death or survival) after severe TBI. Methods: This prospective study enrolled 24 male patients, victims of severe TBI. Venous blood samples were taken in the Intensive Care Unit (ICU) (study entry), 24 and 48 hours later. Plasma cytokine levels were assayed by flow cytometry. Results: Severe TBI was associated with a 42% mortality rate. TBI patients had a significant increase in the levels of all cytokines measured, except for IL-1β, compared to controls. Statistically significant increases in the IL-10, -8 and -6 levels were observed in the non-survivors TBI patients compared to the survivors sub-group measured in the first sample (study entry) and in the subsequent sample (24 hours later). There were no significant differences in IL-1β, TNF-α and IL-12p70 levels between survivors and non-survivors in any time sampled. Conclusions: The findings indicate that increased IL-10, -8 and -6 levels may constitute an early predictor of unfavourable outcome in severe TBI patients.

Development and Validation of a Real-Time Taqman® Polymerase Chain Reaction Assay for the Detection of Mycoplasma gallisepticum in Naturally Infected Birds

Abstract In this study, we report the development and validation of a real-time polymerase chain reaction (PCR) assay using a Taqman®-labeled probe for the detection of Mycoplasma gallisepticum (MGLP assay). The MGLP assay was highly specific with a detection limit of 25 template copies per reaction and a quantification limit of 100 template copies per reaction. Validation of the assay was completed with 1247 samples (palatine cleft and tracheal swabs) from M. gallisepticum-positive and -negative chicken flocks. The MGLP assay was compared to an enzyme-linked immunosorbent assay (ELISA), a conventional polymerase chain reaction assay (mgc2 PCR), and isolation of M. gallisepticum from naturally infected flocks. A total of 805 samples collected from negative flocks, as verified by ELISA and/or mgc2 PCR, were negative by the MGLP assay. A total of 442 samples were collected from positive flocks, of which a total of 228 samples were positive by the MGLP assay. These results agreed for 98.87% of the samples when tested by mgc2 PCR. When comparing the MGLP assay with M. gallisepticum isolation, the MGLP assay was more sensitive than isolation for detecting positive birds from a positive flock, 172/265 and 50/265, respectively. Overall, the MGLP assay and M. gallisepticum isolation agreed for 52.8% of the samples tested. In conclusion, the MGLP assay was highly specific, sensitive, and reproducible, and allowed the quantification of template copies directly from clinical samples.

Elevated Cell-Free Plasma DNA Level as an Independent Predictor of Mortality in Patients with Severe Traumatic Brain Injury

Trauma is the leading cause of death in individuals less than 45 years old worldwide, and up to 50% of trauma fatalities are because of brain injury. Prediction of outcome is one of the major problems associated with severe traumatic brain injury (TBI), and research efforts have focused on the investigation of biomarkers with prognostic value after TBI. Therefore, our aim was to investigate whether cell-free DNA concentrations correlated to short-term primary outcome (survival or death) and Glasgow Coma Scale (GCS) scores after severe TBI. A total of 188 patients with severe TBI were enrolled in this prospective study; outcome variables comprised survival and neurological assessment using the GCS at intensive care unit (ICU) discharge. Control blood samples were obtained from 25 healthy volunteers. Peripheral venous blood was collected at admission to the ICU. Plasma DNA was measured using a real-time quantitative polymerase chain reaction (PCR) assay for the β-globin gene. There was correlation between higher DNA levels and both fatal outcome and lower hospital admission GCS scores. Plasma DNA concentrations at the chosen cutoff point (≥171,381 kilogenomes-equivalents/L) predicted mortality with a specificity of 90% and a sensitivity of 43%. Logistic regression analysis showed that elevated plasma DNA levels were independently associated with death (p<0.001). In conclusion, high cell-free DNA concentration was a predictor of short-term mortality after severe TBI.

Biomarcadores prognósticos no traumatismo crânio-encefálico grave

Trauma is the leading cause of death of people from 1 to 44 years of age. Traumatic brain injury is the main determinant for mortality and morbidity caused by trauma. Outcome prediction is one of the major problems related to severe traumatic brain injury because clinical evaluation has an unreliable predictive value and complicates identification of patients with higher risk of developing secondary lesions and fatal outcome. That is why, there is considerable interest in development of biomarkers that reflect the severity of brain injury and correlate with mortality and functional outcome. Proteins S100B and neuron specific enolases are among the markers most studied for this purpose, however some studies are investigating glial fibrillary acidic protein, creatinine phospokinase, isoenzime B, myelin basic protein, plasma desoxiribonucleic acid, heat shock protein 70, von Willebrand factor, metalloproteinases and brain-derived neurotrophic factor, among others. Evidence suggests that inflammation, oxidative stress, excitotoxicity, neuroendocrine responses and apoptosis play an important role in the development of secondary lesions. Markers involved in these processes are being studied in traumatic brain injury. We reviewed these biomarkers, some of which present promising results for future clinical application.

Prevalence of hepatitis C virus in alcoholic patients: role of parenteral risk factors

BACKGROUND The prevalence of hepatitis C virus (HCV) infection is elevated in alcoholic patients, but the risk factors are unclear. The role of parenteral risk factors are indeterminated in this population. AIMS To determine the prevalence of hepatitis C virus infection in alcoholic patients admitted to a detoxification unit and to evaluate the presence of underlying parenteral risk factors. METHODS A total of 114 consecutive unselected alcoholic patients admitted to a single chemical dependency unit during 14 month were included. Epidemiological data and history of parenteral risk factors for hepatitis C virus infection were obtained with a standardized questionnaire. Blood was collected for determination of aminotransferases and anti-hepatitis C virus antibodies (ELISA-3). Positive samples were confirmed by polymerase chain reaction and tested for genotype. RESULTS Among the 114 alcoholics, 17 (15%) were anti-hepatitis C virus positive. Of these, 12 (71%) had detectable serum HCV-RNA by PCR. Genotype 1 was found in six cases and genotype 3 in five (one patient was undetermined). Forty-nine (43%) patients had elevated serum ALT and/or AST at baseline. The comparison between the 17 positive and the 97 negative patients showed significant differences in mean serum ALT levels (42 +/- 41 IU/L vs. 22 +/- 20 IU/L), rate of elevated ALT (65% vs. 34%), and presence of parenteral risk factors (94% vs. 10%). Comparison between alcoholic patients with and without elevated aminotransferases showed significant difference only in the rate of positive anti-hepatitis C virus antibodies (24% vs. 7%). Furthermore, among the 17 anti-hepatitis C virus positive patients, the rate of detectable HCV-RNA was significantly higher in the 12 with elevated aminotransferases versus the 5 with normal aminotransferases (92% vs. 20%). CONCLUSIONS There was a high prevalence of anti-hepatitis C virus antibodies in alcoholics and the majority was confirmed by the presence of detectable HCV-RNA. Intravenous drug use was the main risk factor for hepatitis C virus infection in this population.

Genotyping of canine distemper virus strains circulating in Brazil from 2008 to 2012

Canine distemper virus (CDV) is a major pathogen of dogs and represents a serious threat to both unvaccinated and vaccinated animals. This study surveyed dogs with or without clinical signs related to canine distemper from different regions of Brazil from 2008 to 2012. A total of 155 out of 386 animals were found to be CDV positive by RT-PCR; 37 (23.8%) dogs were asymptomatic at the time of sampling, and 90 (58%) displayed clinical signs suggestive of distemper. Nineteen (12.2%) dogs had a record of complete vaccination, 15 (9.6%) had an incomplete vaccination protocol, and 76 (49%) had no vaccination record. Based on the sequence analysis of the complete hemagglutinin gene of 13 samples, 12 of the strains were characterized as Genotype South America-I/Europe. Considering criteria of at least 95% nucleotide identity to define a genotype and 98% to define a subgenotype, South America-I/Europe sequences segregated into eight different phylogenetically well-defined clusters that circulated or co-circulated in distinct geographical areas. Together, these findings highlight the relevance of CDV infection in Brazilian dogs, demonstrate the predominance of one genotype in Brazil and support the need to intensify the current control measures.

Molecular Analysis of the Differentiation Potential of Murine Mesenchymal Stem Cells from Tissues of Endodermal or Mesodermal Origin

Mesenchymal stem cells (MSCs) have received great attention due to their remarkable regenerative, angiogenic, antiapoptotic, and immunosuppressive properties. Although conventionally isolated from the bone marrow, they are known to exist in all tissues and organs, raising the question on whether they are identical cell populations or have important differences at the molecular level. To better understand the relationship between MSCs residing in different tissues, we analyzed the expression of genes related to pluripotency (SOX2 and OCT-4) and to adipogenic (C/EBP and ADIPOR1), osteogenic (OMD and ALP), and chondrogenic (COL10A1 and TRPV4) differentiation in cultures derived from murine endodermal (lung) and mesodermal (adipose) tissue maintained in different conditions. MSCs were isolated from lungs (L-MSCs) and inguinal adipose tissue (A-MSCs) and cultured in normal conditions, in overconfluence or in inductive medium for osteogenic, adipogenic, or chondrogenic differentiation. Cultures were characterized for morphology, immunophenotype, and by quantitative real-time reverse transcription-polymerase chain reaction for expression of pluripotency genes or markers of differentiation. Bone marrow-derived MSCs were also analyzed for comparison of these parameters. L-MSCs and A-MSCs exhibited the typical morphology, immunophenotype, and proliferation and differentiation pattern of MSCs. The analysis of gene expression showed a higher potential of adipose tissue-derived MSCs toward the osteogenic pathway and of lung-derived MSCs to chondrogenic differentiation, representing an important contribution for the definition of the type of cell to be used in clinical trials of cell therapy and tissue engineering.

Emergence of a New Genotype of Avian Infectious Bronchitis Virus in Brazil

SUMMARY Infectious bronchitis virus (IBV) is the agent of a highly contagious disease that affects domestic fowl (Gallus gallus). Recent reports showed a high prevalence of one main IBV genotype (Brazil or BR-I) with low genetic diversity in commercial poultry flocks from Brazil. This research analyzed IBV positive poultry flocks from different rearing regions to verify the S1 gene variability and geographic distribution of variant IBV strains in recent years (2010 and 2011). Samples of IBV-positive flocks were obtained from 60 different farms. Forty-nine partial S1 gene sequences were determined and aligned for phylogenetic and amino acid similarity analyses. Eleven samples (22.4%) were similar to Massachusetts vaccine strains (Mass genotype) and 34 samples (69.4%) to the previously characterized Brazilian BR-I genotype. Interestingly, the remaining four samples (8.2%) clustered into a new IBV variant genotype (Brazil-II or BR-II), divergent from the BR-I. A unique nucleotide sequence insertion coding for five amino acid residues was observed in all the Brazilian variant viruses (BR-I and BR-II genotypes). These results show a higher genetic diversity in Brazilian IBV variants than previously described. RESUMEN Aparición de un nuevo genotipo del virus de la bronquitis infecciosa aviar en Brasil. El virus de la bronquitis infecciosa (IBV) es el agente de una enfermedad altamente contagiosa que afecta a las gallinas domésticas (Gallus gallus). Los reportes recientes muestran una alta prevalencia de un genotipo principal de este virus (Brasil o BR-I) con baja diversidad genética en las parvadas avícolas comerciales de Brasil. Esta investigación analiza las parvadas avícolas positivas a la presencia de este virus en diferentes regiones, para verificar la variabilidad del gene S1 y la distribución geográfica de las cepas variantes de este virus en los últimos años (2010 y 2011). Se recolectaron muestras del virus de bronquitis de parvadas positivas en 60 granjas diferentes. Se determinaron 49 secuencias parciales del gene S1 y se alinearon para llevar a cabo análisis filogenéticos y de similitud de aminoácidos. Once muestras (22.4%) fueron similares a la cepa vacunal Massachusetts (Mass) (genotipo Mass) y 34 muestras (69.4%) fueron similares a la cepa brasileña previamente caracterizada, genotipo BR-I. De manera interesante, las cuatro muestras restantes (8.2%) se agruparon en un nuevo genotipo variante (Brasil-II o BR-II), que es divergente de la BR-I. Una inserción única en la secuencia de nucleótidos que codifica para cinco residuos de aminoácidos se observó en todos los virus variantes de Brasil (genotipos BR-I y BR-II). Estos resultados muestran una mayor diversidad genética de las cepas variantes brasileñas del virus de la bronquitis infecciosa a diferencia de lo que se había descrito anteriormente.

Lamivudine resistance and other mutations in the polymerase and surface antigen genes of hepatitis B virus associated with a fatal hepatic failure case

Background and Aim:  Resistance to lamivudine therapy of chronic hepatitis B virus (HBV) infection occurs by mutation in the YMDD motif of the reverse transcriptase (rt) domain (rtM204V/I) of the virus polymerase, and is usually accompanied by rtL180M mutation. Here we investigated virological factors associated with hepatic failure in a 58‐year‐old male, chronically HBV‐infected patient who died after 33 months of lamivudine therapy.