Early derangement of INR predicts liver failure after liver resection for hepatocellular carcinoma.

BACKGROUND: Surgical resection, where appropriate, remains one of the best treatment options for hepatocellular carcinoma (HCC), however outcomes can be compromised by the development of liver failure. We reviewed our experience of liver resection for HCC patients to identify factors that may predict the development of post-hepatectomy liver failure (PHLF) and survival. METHODS: A single centre retrospective cohort study. Data was collected between 1999 and 2017 from all patients undergoing HCC resection in a tertiary university hospital from electronic medical records. PHLF was defined as per the International Study Group for Liver Surgery criteria. Variables with p < 0.15 on univariate analysis were included in a multivariate binary logistic regression model. Kaplan-Meier analyses were used to determine correlations with overall survival (OS) and disease-free survival (DFS), and variables with p < 0.15 on univariate analysis selected for a step-down Cox proportional hazard regression model. RESULTS: Overall, 120 patients underwent liver resection within the study period, of which 22 (18%) developed PHLF. Patients with normal INR ≤1.20 at day 2 did not develop PHLF whereas patients with INR >1.60 were at significant risk. Resection of multiple tumours (odds ratio 21.63, p = 0.002) and deranged postoperative day 2 INR>1.6 (odds ratio 21.05, p < 0.0001) were identified as independent prognostic markers of PHLF. CONCLUSION: The use of INR measurement at day 2 predicts PHLF and may enable us to objectively identify and stratify patients who may be eligible for enhanced recovery programs from those who will merit close monitoring in high dependency areas.

Titer and Distribution of 'Candidatus Phytoplasma pruni' in Prunus avium.

'Candidatus Phytoplasma pruni' infection in cherries causes small, misshapen fruit with poor color and taste, rendering the fruit unmarketable. However, this is a disease with a long development cycle and a scattered, nonuniform symptom distribution in the early stages. To better understand the biology as well as the relationship between pathogen titer and disease expression, we carried out seasonal, spatial, and temporal examinations of 'Ca. P. pruni' titer and distribution in infected orchard-grown trees. Sequential sampling of heavily infected trees revealed marked seasonal patterns, with differential accumulation in woody stem and leaf tissues and, most notably, within fruit in the early stages of development from bloom to pit hardening. Furthermore, mapping phytoplasma distribution and titer in trees at different stages of infection indicated that infection proceeds through a series of stages. Initially, infection spreads basipetally and accumulates in the roots before populating aerial parts of the trees from the trunk upward, with infection of specific tissues and limbs followed by an increasing phytoplasma titer. Finally, we observed a correlation between phytoplasma titer and symptom severity, with severe symptom onset associated with three to four orders of magnitude more phytoplasma than mild symptoms. Cumulatively, these data aid in accurate sampling and management decision-making and furthers our understanding of disease development.

Specific detection of Malus- and Pyrus-infecting viroids by real-time reverse-transcription quantitative PCR assays.

Viroids present a number of issues for their detection and diagnosis because of the absence of symptom expression in many hosts and their low titers in infected plants. However, quarantine programs rely on symptom observations and routine diagnostic testing to reduce the risk of spreading viroid-infected materials to situations where they might affect crop health and production. Sensitive, accurate, and specific assays for viroid detection from both asymptomatic and symptomatic hosts are necessary for managing viroids in post-entry quarantine and certification schemes. The aim of this study was to develop and optimize superior assays based on the reverse-transcription quantitative polymerase chain reaction (RT-qPCR) for the specific detection of apple hammerhead viroid (AHVd), apple scar skin viroid (ASSVd) and pear blister canker viroid (PBCVd). The real-time RT-qPCR assays thus developed detected a greater range of viroid isolates and with greater sensitivity than the current endpoint RT-PCR assays, down to 101 copies per reaction without any amplification of the non-target viroid or virus sequences tested.

Development of RT-qPCR assays for the detection and quantification of three carlaviruses infecting hop.

American hop latent virus (AHLV), hop latent virus (HLV) and hop mosaic virus (HMV) infect members of the Humulus genus worldwide, but very little is known of the biology and etiology of these viruses. A better understanding of these viruses from the molecular level to their economic impact relies on efficient diagnostic assays. Therefore, in this study we developed reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays for the detection of AHLV, HLV, and HMV through an alignment of representative sequences from the National Center for Biotechnology Information (NCBI) database. These assays demonstrated unambiguously their high sensitivity by detecting the respective targets from as low as 102 copies of transcripts per reaction without any amplification from non-targets.

Economic Studies Reinforce Efforts to Safeguard Specialty Crops in the United States.

Pathogen-tested foundation plant stocks are the cornerstone of sustainable specialty crop production. They provide the propagative units that are used to produce clean planting materials, which are essential as the first-line management option of diseases caused by graft-transmissible pathogens such as viruses, viroids, bacteria, and phytoplasmas. In the United States, efforts to produce, maintain, and distribute pathogen-tested propagative material of specialty crops are spearheaded by centers of the National Clean Plant Network (NCPN). Agricultural economists collaborated with plant pathologists, extension educators, specialty crop growers, and regulators to investigate the impacts of select diseases caused by graft-transmissible pathogens and to estimate the return on investments in NCPN centers. Economic studies have proven valuable to the NCPN in (i) incentivizing the use of clean planting material derived from pathogen-tested foundation plant stocks; (ii) documenting benefits of clean plant centers, which can outweigh operating costs by 10:1 to 150:1; (iii) aiding the development of disease management solutions that are not only ecologically driven but also profit maximizing; and (iv) disseminating integrated disease management recommendations that resonate with growers. Together, economic studies have reinforced efforts to safeguard specialty crops in the United States through the production and use of clean planting material.

Development of RT-qPCR assays for the detection of three latent viruses of pome.

Latent fruit tree viruses present economic threat to the industry and nurseries as diseases they cause not only reduce fruit quality and production yield, but can also be spread inadvertently through propagation due to the lack of viral symptoms on an infected mother plant. As a result, these viruses require appropriate detection tools for effective management. In this study we developed RT-qPCR assays for the detection of three latent viruses of pome, apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), and apple mosaic virus (ApMV), using the alignment of representative sequences from the NCBI database. The optimized assays were shown to be specific by successfully amplifying the target from positive controls without showing any detectable amplification in negative and non-target controls, and revealed high sensitivity by reliably detecting as low as 101 copies per reaction. The results also demonstrated that both the choice of extraction method and the reagents used for RT-qPCRcould play a critical role in virus detection outcome. These assays were both reliable and robust compared to the extant RT-PCR methods, and they could be a viable tool for making informed management decisions.

A rational approach to postoperative surveillance for resected non-functional pancreatic neuro-endocrine tumours.

BACKGROUND: Non-functional pancreatic neuroendocrine tumours (NF-PNETs) are rare and have highly variable outcomes. Current guidelines recommend surveillance for NF-PNETs <2 cm. Patients who ultimately have surgical resection are at risk of disease recurrence, and data to support postoperative surveillance protocols are lacking. The aims of this study were to i) identify post-operative predictors of recurrence and ii) risk stratify patients at risk of recurrence. METHODS: Consecutive patients who underwent surgery for NF-PNETs between 2002 and 2015 were identified retrospectively. Data were collected on demographics, pre-operative laboratory results and histopathological tumour characteristics. Statistical analyses were based on penalised Cox-regression modelling and a decision-tree model. Comparison of the variables identified was performed using ROC curves to identify the most sensitive and specific variable associated with disease recurrence. RESULTS: We identified 73 patients (38 males) with a median age of 61.5 years (range: 31-79). The median period of follow-up was 49 months (5-131). During follow up, 10 deaths (13.9%) were recorded and disease recurrence occurred in 12 patients (16.4%). The Kaplan-Meier predicted 1-,3- and 5-year recurrence-free survival rates were 98.6% (95% CI = 95.9, 100%), 85.4% (76.9-94.8%) and 72% (58.7-88.2%) respectively. Cox multivariate analysis identified poor tumour differentiation (WHO G3 grade) and lymph node ratio (LNR) as independent predictors for recurrence (p < 0.05). A simple criterion of 'tumour grade G3 or LNR ≥0.1' was found to be sensitive and specific in detecting disease recurrence. CONCLUSION: Our results have identified a simple and sensitive criterion for risk stratifying post-resection surveillance. Prospective validation in larger patient cohort is now warranted.

Detection and quantification of four viruses in Prunus pollen: Implications for biosecurity.

Pollen transmitted viruses require accurate detection and identification to minimize the risk of spread through the global import and export of pollen. Therefore in this study we developed RT-qPCR assays for the detection of Cherry leaf roll virus (CLRV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Cherry virus A (CVA), four viruses that infect pollen of Prunus species. Assays were designed against alignments of extant sequences, optimized, and specificity was tested against known positive, negative, and non-target controls. An examination of assay sensitivity showed that detection of virus at concentrations as low as 101 copies was possible, although 102 copies was more consistent. Furthermore, comparison against extant assays showed that in both pollen and plant samples, the newly developed RT-qPCR assays were more sensitive and could detect a greater range of isolates than extant endpoint RT-PCR and ELISA assays. Use of updated assays will improve biosecurity protocols as well as the study of viruses infecting pollen.

Development of real-time RT-PCR assays for two viruses infecting pome fruit.

Apple stem grooving virus (ASGV) and Apple green crinkle-associated virus (AGCaV) negatively impact production, maintenance, and distribution of apples and other Malus species world-wide. Due to the increasing diversity of isolates found by high-throughput sequencing, we have developed real-time RT-qPCR assays for these two viruses. Primers and probes were designed against alignments of representative extant sequences from around the world, and reaction conditions optimized for sensitivity and specificity. Assays were validated against a panel of virus isolates, and compared to extant endpoint RT-PCR and ELISA assays. The new real-time RT-qPCR assays showed greater detection sensitivity than extant assays and were able to detect their target viruses from different host tissues.

Bottlenecks and complementation in the aphid transmission of citrus tristeza virus populations.

Aphid transmission is a major factor in the formation of citrus tristeza virus (CTV) populations. Here, we examined the effect of population interaction on aphid transmissibility of different CTV genotypes. We found that there was no correlation between the proportion of viral genotypes in the source population and what was transmitted. We next examined the transmission of a poorly transmitted infectious cDNA clone (T36) in mixture with other CTV genotypes. T36 transmission increased from 0.5% alone, to up to 35.7%, depending on the coinfecting genotype. These results suggest that interaction between CTV genotypes affects the transmission of this virus.

Diversity of three bunya-like viruses infecting apple.

High-throughput sequencing of two trees with apple decline revealed the presence of three bunya-like viruses: apple rubbery wood-associated viruses 1 and 2 (ARWaV-1, ARWaV-2) and citrus concave gum-associated virus (CCGaV), which previously had only been observed in citrus trees. The apple and citrus CCGaV isolates shared over 97% sequence identity. A global collection of apple trees was screened by RT-PCR for these viruses. Twenty-seven of 30 trees were infected with one or more bunya-like virus. Sequence data revealed some diversity among isolates but no geographic grouping. Additional work will be needed to determine if any of these viruses contribute to apple decline.

Isolate fitness and tissue-tropism determine superinfection success.

The mechanism of cross-protection, the deliberate infection of plants with a "mild" virus isolate to protect against "severe" isolates, has long been a topic of debate. In our model system, Citrus tristeza virus (CTV), this appears to be genotype-specific superinfection-exclusion, suggesting a simple recipe for cross-protection. However, this concept failed in field trials, which led us to examine the process of superinfection-exclusion more closely. We found that exclusion relies on the relative fitness of the primary versus the challenge isolates, and the host infected, and that significant differences in superinfection success could occur between isolates that differ by as few as 3 nucleotides. Furthermore, we found that exclusion was not uniform throughout the plant, but was tissue-specific. These data suggest that cross-protection is not a simple like-for-like process but a complex interaction between the primary and challenge isolates and the host.

A real-time RT-qPCR assay for the detection of Citrus tatter leaf virus.

Citrus tatter leaf virus (CLTV) is globally distributed wherever citrus is grown, and, given the extensive use of CTLV sensitive rootstock, has the potential to be a significant threat to the citrus industry. In order to facilitate fast and reliable detection of this virus, we have developed a CTLV-specific real-time RT-qPCR assay. The optimized assay was found to be more reliable and sensitive compared to ELISA and end-point RT-PCR, detecting CTLV in up to 70% more plants. The real-time RT-qPCR is also specific, as it did not cross-react with the closely related Apple stem grooving virus or with the host itself; robust, being able to detect CTLV in young and mature host tissue types; and rapid.

Sequence variation in two genes determines the efficacy of transmission of citrus tristeza virus by the brown citrus aphid.

Vector transmission is an important part of the viral infection cycle, yet for many viruses little is known about this process, or how viral sequence variation affects transmission efficacy. Here we examined the effect of substituting genes from the highly transmissible FS577 isolate of citrus tristeza virus (CTV) in to the poorly transmissible T36-based infectious clone. We found that introducing p65 or p61 sequences from FS577 significantly increased transmission efficacy. Interestingly, replacement of both genes produced a greater increase than either gene alone, suggesting that CTV transmission requires the concerted action of co-evolved p65 and p61 proteins.

Some like it hot: citrus tristeza virus strains react differently to elevated temperature.

Viruses often infect plants as a mixed population. The dynamics of viral populations dictate the success of the infection, yet there is little understanding of the factors that influence them. It is known that temperature can affect individual viruses; could it also affect a virus population? In order to study this, we observed citrus tristeza virus (CTV) populations in different hosts under winter and summer conditions (25 versus 36 °C). We found that only some CTV strains were affected by a higher summer temperature, which lead to a change in CTV population structure, and that this effect was host dependent.

Minor Coat and Heat Shock Proteins Are Involved in the Binding of Citrus Tristeza Virus to the Foregut of Its Aphid Vector, Toxoptera citricida.

Vector transmission is a critical stage in the viral life cycle, yet for most plant viruses how they interact with their vector is unknown or is explained by analogy with previously described relatives. Here we examined the mechanism underlying the transmission of citrus tristeza virus (CTV) by its aphid vector, Toxoptera citricida, with the objective of identifying what virus-encoded proteins it uses to interact with the vector. Using fluorescently labeled virions, we demonstrated that CTV binds specifically to the lining of the cibarium of the aphid. Through in vitro competitive binding assays between fluorescent virions and free viral proteins, we determined that the minor coat protein is involved in vector interaction. We also found that the presence of two heat shock-like proteins, p61 and p65, reduces virion binding in vitro Additionally, treating the dissected mouthparts with proteases did not affect the binding of CTV virions. In contrast, chitinase treatment reduced CTV binding to the foregut. Finally, competition with glucose, N-acetyl-ß-d-glucosamine, chitobiose, and chitotriose reduced the binding. These findings together suggest that CTV binds to the sugar moieties of the cuticular surface of the aphid cibarium, and the binding involves the concerted activity of three virus-encoded proteins. IMPORTANCE: Limited information is known about the specific interactions between citrus tristeza virus and its aphid vectors. These interactions are important for the process of successful transmission. In this study, we localized the CTV retention site as the cibarium of the aphid foregut. Moreover, we demonstrated that the nature of these interactions is protein-carbohydrate binding. The viral proteins, including the minor coat protein and two heat shock proteins, bind to sugar moieties on the surface of the foregut. These findings will help in understanding the transmission mechanism of CTV by the aphid vector and may help in developing control strategies which interfere with the CTV binding to its insect vector to block the transmission.

Finding balance: Virus populations reach equilibrium during the infection process.

Virus populations, mixtures of viral strains or species, are a common feature of viral infection, and influence many viral processes including infection, transmission, and the induction of disease. Yet, little is known of the rules that define the composition and structure of these populations. In this study, we used three distinct strains of Citrus tristeza virus (CTV) to examine the effect of inoculum composition, titer, and order, on the virus population. We found that CTV populations stabilized at the same equilibrium irrespective of how that population was introduced into a host. In addition, both field and experimental observations showed that these equilibria were relatively uniform between individual hosts of the same species and under the same conditions. We observed that the structure of the equilibria reached is determined primarily by the host, with the same inoculum reaching different equilibria in different species, and by the fitness of individual virus variants.

Postoperative CT in pancreas transplantation.

AIM: To examine the usage and value of computed tomography (CT) following simultaneous pancreas and kidney (SPK) transplantation. MATERIALS AND METHODS: Indications for postoperative CT, key findings, and their influence on management were determined by retrospective analysis. RESULTS: Ninety-eight patients underwent 313 CT examinations. Common indications for the examinations included suspected intra-abdominal collection (31.1%) and elevated serum amylase/lipase (24.1%). CT findings most frequently showed non-specific mild inflammation (27.6%), a normal scan (17.1%) and fluid collections (16.3%). High capillary blood glucose (CBG) was associated with resultant CT demonstration of graft vascular abnormalities, but otherwise, particular clinical indications were not associated with specific CT findings. CONCLUSION: Clinical findings in patients with SPK transplants are non-specific. The pattern of abnormalities encountered is significantly different to those seen in native pancreatic disease and demands a tailored protocol. CT enables accurate depiction of vascular abnormalities and fluid collections, thus reducing the number of surgical interventions that might otherwise be required. Elevated CBG should prompt urgent CT to exclude potentially reversible vascular complications.

Anatomical variation and its management in transplantation.

Variant anatomy may be challenging at retrieval, with failure to identify variance being associated with organ damage, particularly vascular damage. On implantation, some variants demand nonstandard techniques of reconstruction or implantation. This review covers the common and less common anatomical variants of the liver, kidney and pancreas, and gives guidance as to how they may be managed during organ retrieval and implantation.

With a little help from my friends: complementation as a survival strategy for viruses in a long-lived host system.

In selective host species, the extent of Citrus tristeza virus (CTV) infection is limited through the prevention of long-distance movement. As CTV infections often contain a population of multiple strains, we investigated whether the members of a population were capable of interaction, and what effect this would have on the infection process. We found that the tissue-tropism limitations of strain T36 in selective hosts could be overcome through interaction with a second strain, VT, increasing titer of, and number of cells infected by, T36. This interaction was strain-specific: other strains, T30 and T68, did not complement T36, indicating a requirement for compatibility between gene-products of the strains involved. This interaction was also host-specific, suggesting a second requirement of compatibility between the provided gene-product and host. These findings provide insight into the 'rules' that govern interaction between strains, and suggest an important mechanism by which viruses survive in a changing environment.