Investigation of the EHV-1 Genotype (N752, D752, and H752) in Swabs Collected From Equids With Respiratory and Neurological Disease and Abortion From the United States (2019-2022).

Contemporary data on equine herpesvirus-1 (EHV-1) genotype (non-neuropathogenic or N752, neuropathogenic or D752 and new variant or H752) in clinically diseased equids is important in order to determine the frequency of these genotypes and their association with disease expression. A total of 297 EHV-1 qPCR-positive swabs collected from 2019 to 2022 from horses with respiratory disease (EHV-1), neurological disease (equine herpesvirus-1 myeloencephalopathy [EHM]) and abortion were tested for the three different EHV-1 genotypes (N752, D752 and H752) using qPCR allelic discrimination assays. All submissions originated from the United States and included 257 EHV-1 cases, 35 EHM cases and 5 cases of abortion. EHV-1 qPCR-positive cases were predominantly seen during winter and spring. N752 was the predominant genotype detected in EHV-1 cases (87.5%), EHM cases (74.3%) and abortions (80%). D752 was detected less frequently in EHV-1 cases (9.3%) and EHM cases (25.7%), while H752 was only detected in EHV-1 cases (3.1%). While the N752 genotype has remained the predominant genotype affecting horses with respiratory disease and abortion, it has also become a leading genotype in cases of EHM, when compared to historical data. The new H752 genotype, first reported in the United States in 2021, has remained confined to a cluster of geographically and temporally related outbreaks and the data showed no emerging spread of H752 since it was first reported. While the monitoring of EHV-1 genotypes is important from a diagnostic and epidemiological standpoint, it may also help establish medical interventions and preventive protocols to reduce the risk of severe complications associated with EHV-1 infection.

A novel approach for simultaneous detection of the most common food-borne pathogens by multiplex qPCR.

Food contaminated with bacterial pathogens is a great threat to human health and food spoilage, having an impact on public health and the food industry. Research in food safety seeks to develop a practical, rapid, and sensitive detection technique for food-borne pathogens. In the past few decades, real-time quantitative polymerase chain reaction (qPCR) has been developed, and multiplex qPCR is a preferred feature. Multiplex qPCR enables the simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. In this study, we have developed and evaluated a hydrolysis (TaqMan) probe-based system for simultaneous detection of eight of the most common food-borne pathogens in a single-step procedure by multiplex qPCR. A multicolor combinational probe coding (MCPC) strategy was utilized that allows multiple fluorophores to label different probes in combinatorial manner. This strategy enabled simultaneous detection, identification, and quantification of targeted genes. The efficiency of the individual qPCR reactions for each target gene had values comparable to those established for multiplex qPCR, with detection limits of approximately < 10 copies of DNA per reaction. Pathogen load helps to predict bacteriological quality status in food products and serves to validate the efficiency of procedures to minimize or eliminate their presence, so newly developed multiplex qPCR was quantitative for each pathogen. During sample preparation, a step to concentrate the target organism from a relatively large sample size, remove all potential PCR inhibitors, and yield samples in a volume suitable for qPCR was incorporated.

Prevalence and geographic distribution of Babesia conradae and detection of Babesia vogeli in free-ranging California coyotes (Canis latrans).

Babesia species are intraerythrocytic piroplasms that can result in disease characterized by hemolytic anemia and thrombocytopenia. Of the 5 species that are known to infect canids in the United States, Babesia conradae is most frequently diagnosed in California, and Babesia vogeli is prevalent in the US. Despite the recent re-emergence of B. conradae, the mechanism of transmission is not known. Coyotes (Canis latrans) have been a proposed reservoir of disease, and previous work has shown that dogs with known aggressive interactions with coyotes are at greater risk for infection. This study aimed to determine the prevalence of B. conradae in wild coyote populations in California to assess the viability of coyotes as a potential source of infection for domestic dogs. Four hundred and sixty-one splenic samples were obtained during post-mortem examination of coyote carcasses from Southern California, Fresno, and Hopland. Demographic data including age, sex, cause of death, and urbanity were collected for each coyote. DNA was extracted from samples and amplified using real-time PCR with primers specific for the B. conradae ITS-2 gene. The 18S gene was amplified and sequenced using conventional PCR primers specific to the Babesia genus from any coyotes positive for B. conradae. In total, 22 coyotes tested positive for B. conradae in Fresno (n = 15), Orange (n = 4), San Bernardino (n = 1), and Los Angeles counties (n = 1) with an overall prevalence of 4.8%. Coyotes from Fresno (P<.01) and rural coyotes (P<.01) were significantly more likely to be infected with B. conradae. Ten of 14 samples sequenced were 99-100% homologous to B. conradae, and 4 samples were 100% homologous with B. vogeli DNA indicating co-infection with both pathogens. This study demonstrates that coyotes can become infected and harbor B. conradae and B. vogeli and should be investigated as a possible source of infection in domestic dogs.

Frequency of Detection of Respiratory Pathogens in Clinically Healthy Show Horses Following a Multi-County Outbreak of Equine Herpesvirus-1 Myeloencephalopathy in California.

Actively shedding healthy horses have been indicated as a possible source of respiratory pathogen outbreak, transmission, and spread. Using nasal swabs from clinically healthy sport horses submitted for qPCR testing after an outbreak of equine herpesvirus-1 (EHV-1) myeloencephalopathy (EHM) in the spring of 2022, this study aimed to identify the rate of clinically healthy horses shedding common and less characterized respiratory pathogens within the sport horse population to better understand their role in outbreaks. Swabs were collected during a required quarantine and testing period, according to the United States Equestrian Federation (USEF), and showed return-to-competition requirements. Common respiratory pathogens, such as equine influenza virus (EIV), EHV-4, and equine rhinitis B virus (ERBV), were found at low but stable frequencies within previously reported ranges, whereas EHV-1 and Streptococcus equi subspecies equi (S. equi) were found at or above previously reported frequencies. Less characterized respiratory pathogens, such as EHV-2, EHV-5, and S. equi subspecies zooepidemicus (S. zooepidemicus), were found within previously reported ranges. Common respiratory pathogens, especially EHV-1 following the multiple EHM outbreaks, were found to be circulating in clinically healthy sport horse populations, reflecting their silent transmission. The strategy of quarantine and EHV-1 qPCR testing of clinically healthy horses was successful at eliminating additional EHM outbreaks and facilitating safe return to competition with no reported respiratory disease outbreaks following the subsequent shows in California.

Relationship between depression and quality of life after myocardial infarction.

Aim To examine the prevalence of depression in patients after acute myocardial infarction (AMI), as well as the relationship between the depression and quality of life. Methods The survey was conducted via sociodemographic questionnaire, Beck Depression Inventory (BDI), and Short Form 36 Health Survey questionnaire (SF-36). The result of SF-36 is expressed in subscales that make up the health status profile, i.e. physical functioning, physical role, emotional role, social functioning, mental health, vitality, pain and general health. Results The study included 120 patients, of which 70 males and 50 females aged between 41 and 88 years (mean 64.73±11.218). All patients were hospitalized at the Clinical Centre of the University of Sarajevo, Clinic for Cardiovascular Disease and Rheumatism, due to complications caused by AMI. After AMI 59 (49.17%) patients had depression. Depression was negatively associated with physical functioning, physical role, emotional role, social functioning, mental health, vitality, pain and general health. Physical functioning (r= -0.701; p<0.01) and physical role (r = -0.538; p<0.01) had the highest correlation with depression. Conclusion The evaluation of depressive symptoms after AMI is imperative, because the appearance of symptoms could have an effect on the patient's quality of life.

Challenges in navigating molecular diagnostics for common equine respiratory viruses.

Equine respiratory viruses remain a leading cause of equine morbidity and mortality, with the resurgence of certain infections, an increasing population of elderly, more susceptible horses, the growth of international equine commerce, and an expansion in geographic distribution of pathogens. The focus of rapid diagnosis of infectious diseases has also shifted recently, with the appearance and increasing importance of nucleic acid amplification-based techniques, primarily polymerase chain reaction (PCR), at the expense of traditional methods such as clinical microbiology. While PCR is fast, reliable, cost-effective, and more sensitive than conventional detection methods, careful interpretation of diagnostic test results is required, taking into account the clinical status of the patient, sample type, assay used and biological relevance of the detected viruses. The interpretation of common equine respiratory viruses such as influenza virus (EIV), alpha herpesviruses (EHV-1, EHV-4), arteritis virus (EAV) and rhinoviruses (ERAV, ERBV) is straight forward as causality can generally be established. However, the testing of less-characterized viruses, such as the gamma herpesviruses (EHV-2, EHV-5), may be confusing, considering their well-established host relationship and frequent detection in both diseased and healthy horses. For selected viruses, absolute quantitation (EHV-1 and EHV-4) and genotyping (EIV and EHV-1) has allowed additional information to be gained regarding viral state and virulence, respectively. This information is relevant when managing outbreaks so that adequate biosecurity measures can be instituted and medical interventions can be considered. The goal of this review is to help the equine practitioner navigate through the rapidly expanding field of molecular diagnostics for respiratory viruses and facilitate the interpretation of results.

Detection of Cephalosporin and Fluoroquinolone Resistance Genes via Novel Multiplex qPCR in Fecal Salmonella Isolates From Northern Californian Dairy Cattle, 2002-2016.

The objectives of this study were to evaluate the prevalence of extended spectrum ß-lactamase (ESBL) genes, AmpC-type ß-lactamase (ACBL) genes, and plasmid mediated quinolone resistance (PMQR) genes in Salmonella isolated at a Veterinary Medical Teaching Hospital microbiology laboratory, examine trends in presence of these resistance genes, and to explore the correlation between phenotypic resistance and presence of specific genes. The presence of ESBL, ACBL, and PMQR genes were detected using a single, novel multiplex qPCR. Only the genes bla CMY-2 and bla TEM were detected in the 110 Salmonella isolates tested. PMQR genes were not detected in isolates screened. Of 94 third-generation cephalosporin resistant isolates, representing eight serotypes, 48% (n = 45) were positive for bla CMY-2 only and 50% (n = 47) were simultaneously positive for bla CMY-2 and bla TEM. Two third-generation cephalosporin resistant isolates were tested negative for all ß-lactamase genes in our qPCR assay and likely house ESBL genes not screened for by our qPCR assay. A logistic regression model revealed that for serotype Dublin isolates (n = 38) the odds ratio for testing positive for bla TEM when compared to all other serotypes was 51.6 (95% CI: 4.01-664.03, p = 0.0029). For serotype Typhimurium (n = 9) the odds ratio for testing positive for bla TEM when compared to all other serotypes was 43.3 (95% CI: 1.76-1000, p = 0.0216). Overall, our results suggest that the prevalence of resistance to cephalosporins and fluoroquinolones due to ESBLs, ACBLs, and PMQR genes present in bovine nontyphoidal Salmonella enterica isolates has remained relatively constant in the isolates screened over a 14-year period.

Recent Pattern of Acute Kidney Injury in Bosnia and Herzegovina.

INTRODUCTION: Acute kidney injury (AKI) is one of the major public health issues with constantly increasing incidence, with epidemiology and outcomes that vary substantially across the world. AIM: Aim of our study was to determine epidemiological characteristics and causes of AKI and to provide a comparison of our findings with data from other low and middle income countries. METHODS: This retrospective observational study conducted during an 18-month period included 84 patients. Data were collected from hospital information system and patients' medical records. All data were analyzed using descriptive statistics. RESULTS: More than two-thirds of patients were older than 56 years. Most cases of AKI (54,76%) were hospital-acquired and predominantly developed in intensive care units (32,14%). Dominant risk factor was underlying chronic kidney disease (48,81%) and chronic heart failure (45,24. In majority of patients (73,81%) were identified multiple factors that may have contributed to AKI: infection (90,48%), prerenal factors (77,38%), nephrotoxic agents (69,05%), and sepsis (28,57%). Multiple organ failure was identified in 94,05% of patients: cardiovascular (64,56%), respiratory (58,23%) and hematological (56,96%) system. Half of all patients were alive at last observation day. Leading cause of death was infection/sepsis (21,43%), followed by cancer (16,67%) and shock (14,28%). CONCLUSION: Data on AKI show great variation, but general picture of AKI resembles more that from high income countries. The need for dialysis and overall mortality remains high. This highlights the importance of early recognition of AKI, timely referral to nephrologist and need for national guidelines and standardized protocols for AKI.

Generality of Post-Antimicrobial Treatment Persistence of Borrelia burgdorferi Strains N40 and B31 in Genetically Susceptible and Resistant Mouse Strains.

A basic feature of infection caused by Borrelia burgdorferi, the etiological agent of Lyme borreliosis, is that persistent infection is the rule in its many hosts. The ability to persist and evade host immune clearance poses a challenge to effective antimicrobial treatment. A link between therapy failure and the presence of persister cells has started to emerge. There is growing experimental evidence that viable but noncultivable spirochetes persist following treatment with several different antimicrobial agents. The current study utilized the mouse model to evaluate if persistence occurs following antimicrobial treatment in disease-susceptible (C3H/HeJ [C3H]) and disease-resistant (C57BL/6 [B6]) mouse strains infected with B. burgdorferi strains N40 and B31 and to confirm the generality of this phenomenon, as well as to assess the persisters' clinical relevance. The status of infection was evaluated at 12 and 18 months after treatment. The results demonstrated that persistent spirochetes remain viable for up to 18 months following treatment, as well as being noncultivable. The phenomenon of persistence in disease-susceptible C3H mice is equally evident in disease-resistant B6 mice and not unique to any particular B. burgdorferi strain. The results also demonstrate that, following antimicrobial treatment, both strains of B. burgdorferi, N40 and B31, lose one or more plasmids. The study demonstrated that noncultivable spirochetes can persist in a host following antimicrobial treatment for a long time but did not demonstrate their clinical relevance in a mouse model of chronic infection. The clinical relevance of persistent spirochetes beyond 18 months following antimicrobial treatment requires further studies in other animal models.

Pathology of coccidioidomycosis in llamas and alpacas.

Coccidioidomycosis is a fungal disease caused by either Coccidioides immitis or Coccidioides posadasii. Anecdotal evidence suggests that camelids are particularly susceptible to this disease and that a relatively large percentage of pneumonias in these animals are caused by Coccidioides spp. In a search of 21 y (1992-2013) of records from the California Animal Health and Food Safety Laboratory, we found 79 cases of coccidioidomycosis diagnosed in camelids; 66 (84%) had pneumonia and 13 (16%) had lesions only in organs other than the lungs. The organs most frequently affected were lung (84%) and liver (78%). Coccidioides spp. were the cause of pneumonia in 66 of 362 (18%) camelid cases during the study period. The lesions in affected organs were multifocal-to-coalescing pyogranulomas, which in most cases were visible grossly. Ten of the 12 formalin-fixed, paraffin-embedded lung samples tested by a universal Coccidioides spp. PCR assay were positive (4 C. immitis, 2 C. posadasii); the species could not be determined in 4 of the 10 cases positive by PCR. Coccidioidomycosis is an important cause of pneumonia in camelids in California, and can be caused by either C. immitis or C. posadasii.

Variable manifestations, diverse seroreactivity and post-treatment persistence in non-human primates exposed to Borrelia burgdorferi by tick feeding.

The efficacy and accepted regimen of antibiotic treatment for Lyme disease has been a point of significant contention among physicians and patients. While experimental studies in animals have offered evidence of post-treatment persistence of Borrelia burgdorferi, variations in methodology, detection methods and limitations of the models have led to some uncertainty with respect to translation of these results to human infection. With all stages of clinical Lyme disease having previously been described in nonhuman primates, this animal model was selected in order to most closely mimic human infection and response to treatment. Rhesus macaques were inoculated with B. burgdorferi by tick bite and a portion were treated with recommended doses of doxycycline for 28 days at four months post-inoculation. Signs of infection, clinical pathology, and antibody responses to a set of five antigens were monitored throughout the ~1.2 year study. Persistence of B. burgdorferi was evaluated using xenodiagnosis, bioassays in mice, multiple methods of molecular detection, immunostaining with polyclonal and monoclonal antibodies and an in vivo culture system. Our results demonstrate host-dependent signs of infection and variation in antibody responses. In addition, we observed evidence of persistent, intact, metabolically-active B. burgdorferi after antibiotic treatment of disseminated infection and showed that persistence may not be reflected by maintenance of specific antibody production by the host.

Single-cell analysis: Advances and future perspectives.

The last several years have seen rapid development of technologies and methods that permit a detailed analysis of the genome and transcriptome of a single cell. Recent evidence from studies of single cells reveals that each cell type has a distinct lineage and function. The lineage and stage of development of each cell determine how they respond to each other and the environment. Experimental approaches that utilize single-cell analysis are effective means to understand how cell networks work in concert to coordinate a response at the population level; recent progress in single-cell analysis is offering a glimpse at the future.

Clinical Validation of a Peritoneal Dialysis Prescription Model in the PatientOnLine Software.

Peritoneal transport characteristics and residual renal function require regular control and subsequent adjustment of the peritoneal dialysis (PD) prescription. Prescription models shall facilitate the prediction of the outcome of such adaptations for a given patient. In the present study, the prescription model implemented in the PatientOnLine software was validated in patients requiring a prescription change. This multicenter, international prospective cohort study with the aim to validate a PD prescription model included patients treated with continuous ambulatory peritoneal dialysis. Patients were examined with the peritoneal function test (PFT) to determine the outcome of their current prescription and the necessity for a prescription change. For these patients, a new prescription was modeled using the PatientOnLine software (Fresenius Medical Care, Bad Homburg, Germany). Two to four weeks after implementation of the new PD regimen, a second PFT was performed. The validation of the prescription model included 54 patients. Predicted and measured peritoneal Kt/V were 1.52 ± 0.31 and 1.66 ± 0.35, and total (peritoneal + renal) Kt/V values were 1.96 ± 0.48 and 2.06 ± 0.44, respectively. Predicted and measured peritoneal creatinine clearances were 42.9 ± 8.6 and 43.0 ± 8.8 L/1.73 m(2)/week and total creatinine clearances were 65.3 ± 26.0 and 63.3 ± 21.8 L/1.73 m(2) /week, respectively. The analysis revealed a Pearson's correlation coefficient for peritoneal Kt/V of 0.911 and Lin's concordance coefficient of 0.829. The value of both coefficients was 0.853 for peritoneal creatinine clearance. Predicted and measured daily net ultrafiltration was 0.77 ± 0.49 and 1.16 ± 0.63 L/24 h, respectively. Pearson's correlation and Lin's concordance coefficient were 0.518 and 0.402, respectively. Predicted and measured peritoneal glucose absorption was 125.8 ± 38.8 and 79.9 ± 30.7 g/24 h, respectively, and Pearson's correlation and Lin's concordance coefficient were 0.914 and 0.477, respectively. With good predictability of peritoneal Kt/V and creatinine clearance, the present model provides support for individual dialysis prescription in clinical practice. Peritoneal glucose absorption and ultrafiltration are less predictable and are likely to be influenced by additional clinical factors to be taken into consideration.

Lyme Borreliosis: Is there a preexisting (natural) variation in antimicrobial susceptibility among Borrelia burgdorferi strains?

The development of antibiotics changed the world of medicine and has saved countless human and animal lives. Bacterial resistance/tolerance to antibiotics have spread silently across the world and has emerged as a major public health concern. The recent emergence of pan-resistant bacteria can overcome virtually any antibiotic and poses a major problem for their successful control. Selection for antibiotic resistance may take place where an antibiotic is present: in the skin, gut, and other tissues of humans and animals and in the environment. Borrelia burgdorferi, the etiological agents of Lyme borreliosis, evades host immunity and establishes persistent infections in its mammalian hosts. The persistent infection poses a challenge to the effective antibiotic treatment, as demonstrated in various animal models. An increasingly heterogeneous subpopulation of replicatively attenuated spirochetes arises following treatment, and these persistent antimicrobial tolerant/resistant spirochetes are non-cultivable. The non-cultivable spirochetes resurge in multiple tissues at 12 months after treatment, with B. burgdorferi-specific DNA copy levels nearly equivalent to those found in shame-treated experimental animals. These attenuated spirochetes remain viable, but divide slowly, thereby being tolerant to antibiotics. Despite the continued non-cultivable state, RNA transcription of multiple B. burgdorferi genes was detected in host tissues, spirochetes were acquired by xenodiagnostic ticks, and spirochetal forms could be visualized within ticks and mouse tissues. A number of host cytokines were up- or down-regulated in tissues of both shame- and antibiotic-treated mice in the absence of histopathology, indicating a lack of host response to the presence of antimicrobial tolerant/resistant spirochetes.

Coinfection with Clostridium piliforme and Felid herpesvirus 1 in a kitten.

A 1-month-old Domestic Shorthair kitten was submitted for autopsy, with a history of upper respiratory tract infection and diarrhea. This was the third kitten from the same litter that had died with similar clinical findings within a period of 1 month. Severe conjunctivitis, rhinitis, tracheitis, and bronchointerstitial pneumonia were present, together with lymphohistiocytic colitis. Respiratory lesions were caused by infection with Felid herpesvirus 1. Colonic lesions were associated with the presence of long filamentous bacteria, identified as Clostridium piliforme, in the cytoplasm of epithelial cells. Our report describes a case of concurrent C. piliforme infection (Tyzzer's disease) and feline rhinotracheitis in a kitten.

A real-time PCR assay for differentiating pathogenic Anaplasma phagocytophilum from an apathogenic, woodrat-adapted genospecies from North America.

Anaplasma phagocytophilum is a tick-transmitted bacterial pathogen of humans and animals comprising strains that cause clinical disease in people, dogs and horses (the pathogenic A. phagocytophilum "genospecies") and more distantly related strains. A rodent-adapted genospecies named DU1, found primarily in woodrats, is unable to infect horses. We developed a real-time PCR (RT-PCR) assay, which targets an 85 base pair region of the ank gene and is specific for the pathogenic genospecies of A. phagocytophilum from North America. Thirty DNA samples from A. phagocytophilum RT-PCR-positive rodents and ticks for which the ank gene had previously been sequenced and had been identified as either the pathogenic genospecies or DU1 were used for validation. All nine samples with the pathogenic genospecies tested positive using the new RT-PCR and all 21 samples with the DU1 genospecies tested negative. Two strains from which the whole genome has been sequenced and are known to be pathogenic (A. phagocytophilum, strain HZ isolated from a human from New York and strain MRK isolated from a horse from California) both tested positive using the new RT-PCR assay. We also tested blood from 20 horses and six dogs that were RT-PCR-positive using a previously validated RT-PCR protocol: all were PCR-positive using the new assay as well. The assay has high efficiency and reproducibility and there was no cross-reaction with related and tick-borne bacteria tested, although the assay cross-reacts with the ungulate-adapted genospecies Ap-Variant 1. This new RT-PCR assay will aid in future research on the ecology and epidemiology of A. phagocytophilum by allowing researchers to easily identify the pathogenic genospecies in reservoir hosts and vectors.

Incidence of subclinical hypothyroidism in renal transplant patients.

UNLABELLED: Thyroid disorders are common in chronic kidney disease. THE AIM: The purpose of this study was to compare thyroid gland disorders among healthy participants and renal transplant patients and to assess the duration of dialysis on thyroid disorders before transplantation. MATERIAL AND METHODS: Prospective study during 12 months period included 80 participants divided into two groups. Study group of 40 patients with transplanted kidney was divided in two subgroups, according to the time spent on dialysis (i.e. under and over 72 months). The control group included 40 healthy participants. The exclusion criteria was represented by the previous thyroid disorders and systemic illnesses and treatment with drugs that interfere with thyroid function (amiodarone, propranolol, lithium). The blood samples were taken for standard laboratory analysis, total thyroid hormone levels. Serum level of free thyroxine (T4) and free triiodothyronine (T3) were assayed by RIA using commercially available kits. Subclinical hypothyroidism is defined by the finding of elevated thyroid-stimulating hormone (TSH) > 4.4 mmol/L and normal values of T3 and T4. RESULTS: The relative distribution of the functional thyroid disorders is statistically significantly higher in the experimental group: the low T3 syndrome in 12.5% (n = 5) patients (p = 0.017); low T4 syndrome in 7.5% (n = 3) patients (p = 0.072) and subclinical hypothyroidism in 17.5% (n = 7) patients (p = 0.021). There is statistically significant difference in the relative representation (percentage) between respondents to 72 months and respondents over 72 months duration of hemodialysis, namely: low T3 syndrome, which is a higher percentage was recorded in patients up to 72 months duration of dialysis (19.23%), then subclinical hypothyroidism where a greater percentage recorded in subjects over 72 months duration of dialysis (35.71%) before transplantation. CONCLUSION: Considering that we are found in kidney transplant patients a significant link of subclinical hypothyroidism with decreased level of T3 and higher incidence of low T3 syndrome, which are associated with increased cardiovascular mortality and morbidity, and act as markers of survival patients after transplantation, it is necessary to conduct a periodically measuring levels of T3, T4 and TSH in these patients in order to assess the relationship between thyroid dysfunction and mortality risk in this population.

Development and validation of a multiplex reverse transcription quantitative PCR (RT-qPCR) assay for the rapid detection of Citrus tristeza virus, Citrus psorosis virus, and Citrus leaf blotch virus.

A single real-time multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay for the simultaneous detection of Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus leaf blotch virus (CLBV) was developed and validated using three different fluorescently labeled minor groove binding qPCR probes. To increase the detection reliability, coat protein (CP) genes from large number of different isolates of CTV, CPsV and CLBV were sequenced and a multiple sequence alignment was generated with corresponding CP sequences from the GenBank and a robust multiplex RT-qPCR assay was designed. The capacity of the multiplex RT-qPCR assay in detecting the viruses was compared to singleplex RT-qPCR designed specifically for each virus and was assessed using multiple virus isolates from diverse geographical regions and citrus species as well as graft-inoculated citrus plants infected with various combination of the three viruses. No significant difference in detection limits was found and specificity was not affected by the inclusion of the three assays in a multiplex RT-qPCR reaction. Comparison of the viral load for each virus using singleplex and multiplex RT-qPCR assays, revealed no significant differences between the two assays in virus detection. No significant difference in Cq values was detected when using one-step and two-step multiplex RT-qPCR detection formats. Optimizing the RNA extraction technique for citrus tissues and testing the quality of the extracted RNA using RT-qPCR targeting the cytochrome oxidase citrus gene as an RNA specific internal control proved to generate better diagnostic assays. Results showed that the developed multiplex RT-qPCR can streamline viruses testing of citrus nursery stock by replacing three separate singleplex assays, thus reducing time and labor while retaining the same sensitivity and specificity. The three targeted RNA viruses are regulated pathogens for California's mandatory "Section 3701: Citrus Nursery Stock Pest Cleanliness Program". Adopting a compatible multiplex RT-qPCR testing protocol for these viruses as well as other RNA and DNA regulated pathogens will provide a valuable alternative tool for virus detection and efficient program implementation.

Posterior reversible encephalopathy syndrome (PRES) as a complication of immunosuppressive therapy in renal transplantation in children.

Although kidney transplantation is by far the best method of renal replacement therapy, organ receiver is still not spared of eventual toxic consequences of drugs that are in charge of keeping the transplanted kidney functional. Both calcineurin inhibitors, of which tacrolimus more often, occasionally lead to neurotoxic side effects, mostly mild and reversible and dose-dependent in nature, but they can also be very severe or even fatal. It is very important to be aware of possible neurotoxic effects, to confirm them radiologically, and to prevent or reduce drug effects on nervous system. Sometimes the reduction of dose or substitution with another drug with similar mechanism effect is sufficient to terminate the neurotoxic effects of the drug and still not jeopardize the function of transplanted organ.

Methylation and gene expression responses to ethanol feeding and betaine supplementation in the cystathionine beta synthase-deficient mouse.

BACKGROUND: Alcoholic steatohepatitis (ASH) is caused in part by the effects of ethanol (EtOH) on hepatic methionine metabolism. METHODS: To investigate the phenotypic and epigenetic consequences of altered methionine metabolism in this disease, we studied the effects of 4-week intragastric EtOH feeding with and without the methyl donor betaine in cystathionine beta synthase (CßS) heterozygous C57BL/6J mice. RESULTS: The histopathology of early ASH was induced by EtOH feeding and prevented by betaine supplementation, while EtOH feeding reduced and betaine supplementation maintained the hepatic methylation ratio of the universal methyl donor S-adenosylmethionine (SAM) to the methyltransferase inhibitor S-adenosylhomocysteine (SAH). MethylC-seq genomic sequencing of heterozygous liver samples from each diet group found 2 to 4% reduced methylation in gene bodies, but not promoter regions of all autosomes of EtOH-fed mice, each of which were normalized in samples from mice fed the betaine-supplemented diet. The transcript levels of nitric oxide synthase (Nos2) and DNA methyltransferase 1 (Dnmt1) were increased, while those of peroxisome proliferator receptor-α (Pparα) were reduced in EtOH-fed mice, and each was normalized in mice fed the betaine-supplemented diet. DNA pyrosequencing of CßS heterozygous samples found reduced methylation in a gene body of Nos2 by EtOH feeding that was restored by betaine supplementation and was correlated inversely with its expression and positively with SAM/SAH ratios. CONCLUSIONS: The present study has demonstrated relationships among EtOH induction of ASH with aberrant methionine metabolism that was associated with gene body DNA hypomethylation in all autosomes and was prevented by betaine supplementation. The data imply that EtOH-induced changes in selected gene transcript levels and hypomethylation in gene bodies during the induction of ASH are a result of altered methionine metabolism that can be reversed through dietary supplementation of methyl donors.