Automation and standardisation of clinical molecular testing using PCR.Ai - A comparative performance study.

BACKGROUND: We undertook a prospective clinical study to evaluate PCR.Ai's (www.pcr.ai) accuracy and impact when automating the manual data-analysis and quality control steps associated with routine clinical pathogen testing using real-time PCR (qPCR). OBJECTIVES: We evaluated the impact of PCR.Ai when used as the final interpretation/verification step for routine in-house qPCR tests for respiratory pathogens and for norovirus for a total of 22,200 interpretations. STUDY DESIGN: We compared PCR.Ai to our existing manual interpretation, to determine accuracy and hands-on time savings. PCR.Ai was accurate. RESULTS AND CONCLUSIONS: There was 100% concurrence between validated respiratory virus and norovirus detection by our manual routine analysis method and PCR.Ai. Furthermore, there were significant routine savings with PCR.Ai of 45 min/respiratory run and 32 min/norovirus run. Our conclusion is that PCR.Ai is a highly accurate time-saving tool that reduces complexity of qPCR analysis and hence the need for specialists and hands-on time. It demonstrated capabilities to enable us to get results out more quickly with lower costs and less risk of errors.

Ileocaecal junction carcinoma: a clinicopathological study of 199 cases.

AIM: The ileocaecal junction (ICJ) region is an epithelial transition zone in which carcinomas are frequently diagnosed. However, it is currently unknown whether ICJ carcinomas (ICJ-CAs) have distinctive features. This study aimed to characterize the clinicopathological features of ICJ-CAs. METHOD: All ileal and colorectal resections for carcinoma, performed in Calgary, Canada between January 2009 and June 2012, were reviewed. Carcinomas in which the epicentre was within 5 cm of the ileocaecal valve (ICV) were defined as ICJ-CAs. Of 1003 carcinomas studied, 199 (19.8%) were ICJ-CAs, including 93 (9.3%) that crossed the ICV. Comparison of clinicopathological features with carcinomas of the other ileo-colorectal regions was made. Survival was also assessed. RESULTS: Clinically, ICJ-CAs were more common in female than male patients (56.3% female) compared with left-colonic (42.9% female) and rectal (37.9% female) carcinomas, and were more common in older age-groups of patients (71.8 ± 12.7 years) compared with appendiceal (62.6 ± 11.3 years), left-colonic (69.4 ± 12.3 years) and rectal (67.1 ± 11.9 years) carcinomas. Macroscopically, ICJ-CAs were similar to other colorectal carcinomas and were mostly described as ulcerated (63.3%). Histologically, ICJ-CAs had more mucinous, signet-ring cell and/or neuroendocrine features (39.7%, 8.0% and 7.5%, respectively) than did carcinomas of the left colon (16.8%, 1.6% and 1.1%, respectively) and the rectum (14.1%, 1.0% and 0.0%, respectively). They were higher grade (20.1% were high grade) than those of the left-colon (10.3%) and the rectum (9.8%). ICJ-CAs presented at a higher T-stage (25.6% were T4) compared with rectal carcinomas (11.6%). Most significantly, ICJ-CAs presented at a higher N-stage (25.6% were N2) than did right-colonic (14.1%) and rectal (16.2%) carcinomas. Although survival of patients with ICJ-CAs did not differ from those with right-colonic carcinomas, those with carcinomas directly involving the ICV did show a significantly decreased survival. CONCLUSION: ICJ-CAs display several distinct clinicopathological features that may require special diagnostic, prognostic and management attention.

One compared with two cycles of mitomycin C in chemoradiotherapy for anal cancer: analysis of outcomes and toxicity.

BACKGROUND: Concurrent chemoradiation with fluorouracil (5fu) and mitomycin C (mmc) is standard treatment for anal canal carcinoma (acc). The current protocol in Alberta is administration of 5fu and mmc during weeks 1 and 5 of radiation. However, administration of the second bolus of mmc has been based largely on centre preference. Given limited published data on outcomes with different mmc regimens, our objective was to compare the efficacy and toxicity of 1 compared with 2 cycles of mmc in acc treatment. METHODS: Our retrospective study evaluated 169 acc patients treated with radical chemoradiotherapy between 2000 and 2010 at two tertiary cancer centres. All patients were treated with 2 cycles of 5fu and with 1 cycle (mmc1) or 2 cycles (mmc2) of mmc. Acute toxicities, disease-free (dfs) and overall survival (os) were analyzed. RESULTS: Baseline demographics, performance status, and stage were similar in the groups of patients who received mmc1 (52%) and mmc2 (48%). Before treatment, median hematologic parameters were comparable, except for white blood cell count, which was higher in the mmc2 group, but within normal range. The 5-year os and dfs were similar (75.1% and 54.2% for mmc1 vs. 70.7% and 44.2% for mmc2, p = 0.98 and p = 0.63 respectively). On multivariate analysis, mmc2 was the factor most strongly associated with specific acute toxicities: grade 3+ leukopenia (hazard ratio: 4.82; p < 0.01), grade 3+ skin toxicity (hazard ratio: 4.76; p < 0.001), and hospitalizations secondary to febrile neutropenia (hazard ratio: 9.91; p = 0.001). CONCLUSIONS: In definitive chemoradiotherapy for acc, 1 cycle of mmc appears to offer outcomes similar to those achieved with 2 cycles, with significantly less acute toxicity.

Simultaneous detection and quantitation of cytomegalovirus, Epstein-Barr virus, and adenovirus by use of real-time PCR and pooled standards.

Quantitative real-time PCR has become the most widely used preemptive approach for managing cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus infections in immunosuppressed patients. These three assays are normally available as separate tests, each using five quantitation standards that are tested in duplicate. We have developed an adenovirus-CMV-EBV triplex assay that uses one set of five pooled quantitative standards, tested singly rather than in duplicate. This test demonstrated a sensitivity and an accuracy of quantitation equivalent to those of our previous single tests and was shown to be able to detect mixed infections with no loss in sensitivity. This assay is now in routine use in our laboratory and has considerably simplified the work flow of the laboratory, with a resultant improvement in sample turnaround time and significantly reduced costs.

Outcome of patients undergoing liver transplantation for primary sclerosing cholangitis.

PURPOSE: The purpose of this study was to determine the outcome of patients with inflammatory bowel disease who underwent liver transplantation for primary sclerosing cholangitis. METHODS: All patients who underwent liver transplantation for primary sclerosing cholangitis at our institution were identified. A review of patients' hospital and office charts was performed; all patients were then contacted, and a detailed survey was administered by telephone. RESULTS: Sixty-nine patients were identified. There were 53 males (76.8 percent) and 16 females, with a mean age of 45.3 (+/- 13.3) years. Fifty-two (75.4 percent) of the 69 patients had documented inflammatory bowel disease; of these, 40 had ulcerative colitis (76.9 percent), 11 had Crohn's disease, and 1 had indeterminate colitis. Thirty-one patients (60 percent) were diagnosed with inflammatory bowel disease before primary sclerosing cholangitis, with a mean interval to diagnosis of primary sclerosing cholangitis of 10.8 (+/- 10.3) years. Seven patients had both diagnoses made at roughly the same time, and 14 patients initially were diagnosed with primary sclerosing cholangitis and subsequently were found to have inflammatory bowel disease, with a mean interval of 5.2 (+/- 4.4) years; 5 (35.7 percent) of those 14 patients were only diagnosed with inflammatory bowel disease after their liver transplant. The mean time from diagnosis of primary sclerosing cholangitis to liver transplantation was 6.1 (+/- 4.9) years. Since their transplant, 30.8 percent of patients rated their colitis as worse, 38.5 percent felt it was unchanged, and 30.8 percent felt that their colitis was better controlled. Eight (15.4 percent) of the 52 patients with inflammatory bowel disease denied having any knowledge of an increased risk of colorectal neoplasia. Four patients have required colectomy for colorectal neoplasia after liver transplantation, at a mean of 4.7 years after transplantation. Of the patients with inflammatory bowel disease, 42 (80.1 percent) had at least 1 posttransplant surveillance colonoscopy. Eight of the remaining ten patients had a colectomy, leaving only two patients (3.8 percent) who had not been surveyed. However, only 32 (61.5 percent) of the patients with inflammatory bowel disease have been on a surveillance regimen that would approximately conform to current screening recommendations. CONCLUSIONS: The activity of inflammatory bowel disease after transplantation is highly variable. Patients appeared to lack knowledge of their increased risk for colorectal neoplasia. Colorectal cancer is an uncommon but important complication in patients after liver transplantation for primary sclerosing cholangitis, and ongoing surveillance is required. Patients may require education to increase their awareness of the cancer risk and compliance with surveillance.

Consent, sectionalisation and the concept of a medical procedure.

Consent transforms an otherwise illegitimate act into a legitimate one. To be valid, however, it must be adequately informed. The legal requirement is vague and provides little assistance in predicting when it will be satisfied. This is particularly so when a patient consents to a procedure and the physician subsequently varies one of the components of that procedure. Using three legal judgments and one General Medical Council (GMC) decision as a springboard, I have explored the concept of a medical procedure within the context of consent and developed a theoretical model to elucidate a more predictable and consistent informational requirement.

Replication of the herpes simplex virus type 1 RL1 mutant 1716 in primary neuronal cell cultures--possible relevance to use as a viral vector.

The herpes simplex virus type 1 (HSV-1) RL1 deletion mutant 1716 has properties that make it a promising candidate as a viral vector for gene therapy in the human nervous system. These properties include its ability to spread along neural pathways and establish a latent infection in post-mitotic neurons, while retaining a non-virulent phenotype in vivo and an inability to cause a lytic infection in stationary or fully differentiated cells. In this study, we used viral replication assays and indirect immunofluorescence to investigate the ability of 1716 to bind to, enter, express genes and produce progeny virus in dissociated neuronal cell cultures prepared from rat hippocampal, medial septal and dorsal root ganglion (DRG) tissues and in primary rat astrocyte cultures. Both heterogeneous cultures and those that had been enriched for neurons were employed. Following both low and high multiplicities of virus infection, the behaviour of 1716 was compared with its wild-type parent HSV-1 strain 17 in these cultures. It was found that the growth of 1716 was significantly impaired compared to wild type HSV-1, with these differences being magnified at lower multiplicities of viral infection as well as in neuron-enriched cultures: this impairment is likely to be due to decreased replication, as immunofluorescence assays showed that 1716 bound to, entered and expressed genes in all neuronal cell types and astrocytes with similar efficiency to the wild type virus. This ability of 1716 to enter and express genes in different neuronal populations demonstrates its potential suitability as a viral vector.

The Human Rights Act 1998 and the individual's right to treatment.

The right to demand treatment--even when life-saving--is not recognised by English common law. The courts have consistently stated that they do not have the jurisdiction to order a doctor to perform a particular treatment. This article considers whether the impending Human Rights Act 1998 can be interpreted so as to allow this right. While a general right to treatment is discussed the argument focuses on life-saving treatment. As an illustration, the David Glass case will be analysed and the impact of the Human Rights Act will be examined by considering how the judgment might have differed had the Act been in force.

Herpes simplex virus type 1 gene UL14: phenotype of a null mutant and identification of the encoded protein.

Herpes simplex virus type 1 (HSV-1) gene UL14 is located between divergently transcribed genes UL13 and UL15 and overlaps the promoters for both of these genes. UL14 also exhibits a substantial overlap of its coding region with that of UL13. It is one of the few HSV-1 genes for which a phenotype and protein product have not been described. Using mass spectrometric and immunological approaches, we demonstrated that the UL14 protein is a minor component of the virion tegument of 32 kDa which is expressed late in infection. In infected cells, the UL14 protein was detected in the nucleus at discrete sites within electron-dense nuclear bodies and in the cytoplasm initially in a diffuse distribution and then at discrete sites. Some of the UL14 protein was phosphorylated. A mutant with a 4-bp deletion in the central region of UL14 failed to produce the UL14 protein and generated small plaques. The mutant exhibited an extended growth cycle at low multiplicity of infection and appeared to be compromised in efficient transit of virus particles from the infected cell. In mice injected intracranially, the 50% lethal dose of the mutant was reduced more than 30,000-fold. Recovery of the mutant from the latently infected sacral ganglia of mice injected peripherally was significantly less than that of wild-type virus, suggesting a marked defect in the establishment of, or reactivation from, latent infection.

Histopathological responses in the CNS following inoculation with a non-neurovirulent mutant (1716) of herpes simplex virus type 1 (HSV 1): relevance for gene and cancer therapy.

The RL1 gene of herpes simplex virus (HSV) encodes a polypeptide, ICP34.5 which is a specific virulence determinant. RL1 null mutants fail to replicate in both the PNS and CNS and are incapable of causing encephalitis. Additionally, RL1 null mutants have the capacity to replicate in actively dividing cells but fail to replicate in growth arrested or terminally differentiated cells. This selective replication phenotype has highlighted their use as both tumour killing agents and gene delivery vehicles particularly to the nervous system. Before their full potential can be assessed, however, it is necessary to determine the pathological and immune responses induced following direct intracerebral inoculation. Fourteen mice were injected in the left cerebral hemisphere with a high dose of the HSV-1, RL1 null mutant 1716. At regular time intervals up to 28 days, the mice were killed and the distribution of virus antigen, histopathological changes and immune responses in the CNS determined by H & E staining and immunohistochemistry. Control mice were injected with either wild type HSV-1 or buffer. At early times post-inoculation with 1716, there is a low grade meningoencephalitis with a limited inflammatory response. This is accompanied by virus antigen expression confined to the site of inoculation. By 28 days the CNS is histopathologically normal; virus antigen and immune responses are no longer detectable. These findings demonstrate that infection of the CNS by RL1 null mutants of HSV results in a finite, self-limiting response and highlights their potential for therapeutic use.

Functional analysis of the herpes simplex virus type 2 strain HG52 RL1 gene: the intron plays no role in virulence.

Sequence analysis predicts that herpes simplex virus type 2 (HSV-2) strain HG52 contains an open reading frame, RL1, encoding a polypeptide equivalent to ICP34.5 of HSV-1. Similarly to HSV-1, deletion of the region spanning RL1 abolishes the virulence of HSV-2 strain HG52 and its ability to grow in stationary 3T6 cells. In contrast to HSV-1, the HSV-2 strain HG52 RL1 gene is predicted to contain a 154 bp intron. Previously, we have demonstrated that this intron is spliced from RL1 poly(A)+ mRNA at the predicted splice donor/ acceptor sites. To determine if the intron affects the function of ICP34.5 of HSV-2 strain HG52, we have constructed a virus, 2624, in which the RL1 intron is deleted: 2624 retains wild-type growth both in vivo and in 3T6 cells, indicating that the presence of an intron does not affect the function of RL1 in HSV-2 strain HG52. 2624 has wild-type growth kinetics in BHK21/C13 cells.

High efficiency gene transfer to the central nervous system of rodents and primates using herpes virus vectors lacking functional ICP27 and ICP34.5.

The safe and efficient use of herpes simplex virus (HSV)-based vectors to deliver genes of potentially therapeutic benefit to the central nervous system will require their effective disablement by the inactivation of viral genes required for lytic growth. Here we report that viruses lacking functional genes for ICP27 (which is required for growth in all cell types) and ICP34.5 (which is required for growth in nondividing cell types) can deliver a marker gene to both the rodent and primate CNS with high efficiency whilst producing relatively minimal damage and having no effect on sodium currents in dorsal root ganglion neurons. Such viruses paradoxically deliver genes at much higher efficiency than the less disabled single mutant lacking ICP34.5 alone and also, as expected, produce less damage in vivo. Moreover, unlike the single mutant lacking ICP27 the double mutant viruses cannot revert to wild-type by acquistion of complimenting gene sequences during growth of virus stocks in vitro on dividing cells expressing ICP27 since artificial expression of ICP34.5 in these cells is not required. Such ICP27-; ICP34.5- viruses thus offer a platform for the development of vectors which are sufficiently safe for ultimate use in human gene therapy.

Preparation of HSV-DNA and Production of Infectious Virus.

This chapter deals with (1) the preparation of herpes simplex virus (HSV) virion DNA of a quality and purity suitable to be used for the generation of infectious virus, and (2) its use in the preparation of infectious virus. An important development in the understanding of virus genetics and gene products has been the ability to carry out reverse genetics. This is dependent on the ability to manipulate the genome in vitro and reconstitute infectious virus. Our understanding of DNA viruses and positive stranded RNA viruses (where DNA and RNA/cDNA, respectively, are generally infectious) is considerably greater than for negative stranded RNA viruses, where until recently, it had been impossible to generate virus.from either RNA or cDNA. Within the herpesviridae, knowledge of the function of HSV gene products is one of the more advanced owing to the relatively straightforward techniques required to generate virus from HSV-DNA, and to introduce desired mutations by cloning small parts of the genome, manipulating them, and then reintroducing the mutations by a process of cotransfection and in vivo recombination with intact virus DNA. Other α-herpesviruses, such as EHV-1 and PRV, are equally amenable to such manipulation, and knowledge of their gene products is also well advanced. In contrast, this technology is only now, and with much less success, being applied to other members of the family, such as EBV, HCMV, and VZV, and knowledge of their genetics is much less advanced. The use of cosmids to reconstitute intact virus will aid in the advance of knowledge for these viruses. For examples of uses of recombinant DNA technology, the reader is referred to other chapters (especially those on cloning and mutagenesis). I will concentrate on the techniques currently in use in my laboratory, but will also mention other techniques in use elsewhere that may be more appropriate in certain cell types.

The herpes simplex virus virulence factor ICP34.5 and the cellular protein MyD116 complex with proliferating cell nuclear antigen through the 63-amino-acid domain conserved in ICP34.5, MyD116, and GADD34.

The herpes simplex virus (HSV) virulence factor ICP34.5, the mouse myeloid differentiation protein MyD116, and the hamster growth arrest and DNA damage protein GADD34 share a 63-amino-acid carboxyl domain which has significant homologies to otherwise divergent proteins. Here we report that both ICP34.5 and its cellular homolog MyD116 complex through the conserved domain with proliferating cell nuclear antigen. In addition, HSV infection induces a novel 70-kDa cellular protein detectable by antisera to both ICP34.5 and GADD34, demonstrating that this novel protein possesses homology with the 63-amino-acid conserved domain.

Use of a "replication-restricted" herpes virus to treat experimental human malignant mesothelioma.

Modified, nonneurovirulent herpes simplex viruses (HSVs) have shown promise in the treatment of brain tumors. However, HSV-1 can infect and lyse a wide range of cell types. In this report, we show that HSV-1716, a mutant lacking both copies of the gene coding ICP-34.5, can effectively treat a localized i.p. malignancy. Human malignant mesothelioma cells supported the growth of HSV-1716 and were efficiently lysed in vitro. i.p. injection of HSV-1716 into animals with established tumor nodules reduced tumor burden and significantly prolonged survival in an animal model of non-central nervous system-localized human malignancy without dissemination or persistence after i.p. injection into SCID mice bearing human tumors. These findings suggest that this virus may be efficacious and safe for use in localized human malignancies of nonneuronal origin such as malignant mesothelioma.

Gene delivery to rat enteric neurons using herpes simplex virus-based vectors.

Neurons of the enteric (gut) nervous system can be cultured in vitro and readily survive transplantation into the brain making close connections with host neurons. As such, they could potentially be used to deliver therapeutic gene products to the brain after transduction with appropriate genes in culture. Here the authors report the first example of gene delivery to such cultured neurons using herpes simplex virus based vectors. They show that viruses lacking the immediate early gene encoding ICP27 (which are unable to replicate lytically) can efficiently deliver a marker gene to enteric neurons without producing extensive cellular damage. In contrast, viruses lacking only the viral neurovirulence factor encoded by ICP34.5 are inefficient in gene delivery, and produce extensive cellular damage, although they cannot replicate lytically in enteric neurons. A virus lacking both ICP27 and ICP34.5, however, produces less cellular damage than one lacking only ICP27, and is as efficient in gene transfer, whereas inactivation of VMW65 reduces toxicity further. The identification of this virus as a safe and efficient gene delivery vector for enteric neurons paves the way for the eventual delivery of therapeutic genes and subsequent transplantation of engineered neurons into the CNS.

Gene delivery to the central and peripheral nervous systems of mice using HSV1 ICP34.5 deletion mutant vectors.

Herpes simplex virus 1 (HSV1) ICP34.5 deletion mutants are avirulent upon inoculation of both the central and peripheral nervous systems of mice, but they replicate to near wild-type titres in a number of non-neuronally derived cell lines in culture. Thus these mutants might be suitable for development as safe vectors for gene transfer to the nervous system. However, the mechanism of this avirulent phenotype in neuronal cells is at present poorly understood, although it has been suggested that nonpermissive cells infected with these mutants may undergo apoptosis, the function of ICP34.5 being to prevent this response and to allow continued virus replication. If this were the case ICP34.5 null mutants might be unsuitable for gene transfer as infected cells would quickly die, limiting the expression of a transgene. Here we have inserted a beta-galactosidase marker gene into a nonessential gene of an HSV1 strain 17+ mutant in which ICP34.5 has been deleted and also into a second mutant in which the virion transactivator protein VMW65 is also inactive. While all the mutants grew to high titre in tissue culture, mice inoculated by the foot-pad or intracranial route at high titre remained healthy until the end of the experiment. Moreover, beta-galactosidase was expressed either in the brain or in the dorsal root ganglia, depending on the site of inoculation. This suggests that in vivo the absence of ICP34.5 does not prevent the expression of a transgene in neuronal tissue and indicates that non-neurovirulent mutants lacking this gene may be suitable for further development as safe vectors for gene therapy in vivo.