RESUMO
BACKGROUND AND AIMS: The DRAINAGE trial was a randomized controlled trial comparing preoperative endoscopic (EBD) and percutaneous biliary drainage (PTBD) in patients with potentially resectable, perihilar cholangiocarcinoma (pCCA). The aim of this study was to compare the long-term outcomes. METHODS: Patients were randomized in four tertiary referral centers. Follow-up data were available for all included patients. Primary outcome was overall survival (OS). Secondary outcomes were readmissions, and re-interventions not including in-trial interventions. RESULTS: A total of 54 patients were randomized; 27 in both groups. Median follow-up for both groups was 62 months (95% CI 54-70). The median OS was 13 months (95% CI 7.9-18.1) in the EBD and 7 months (95% CI 0.0-17.2) in the PTBD group (P = 0.28). Twenty (37%, n = 8 EBD vs n = 12 PTBD, P = 0.43) of 54 patients were readmitted at least once, mostly due to drainage-related complications (n = 13, 24%). Of note, 14 out of the 54 patients died within the trial. A total of 76 drainage procedures (32 EBD and 44 PTBD) were performed in 28 patients. The median number of stent or drain placements was 2 (2-4) for the EBD group and 2 (1-3) for the PTBD group (P = 0.77). DISCUSSION: Although this follow-up study represented a small cohort, no long-term differences in survival, readmissions, and drainage procedures for EBD and PTBD were found, even when comparing the resected and unresected group. However, this study demonstrates the complexity of biliary drainage for patients with potentially resectable pCCA, even in tertiary referral centers.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Tumor de Klatskin , Humanos , Tumor de Klatskin/patologia , Seguimentos , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/cirurgia , Drenagem/efeitos adversos , Ductos Biliares Intra-Hepáticos/cirurgiaRESUMO
BACKGROUND: Biliary tract cancer (BTC) is an uncommon cancer with an unfavorable prognosis. Since 2010, the standard of care for patients with unresectable BTC is palliative treatment with gemcitabine plus cisplatin, based on the landmark phase III ABC-02 trial. This current study aims to evaluate the efficacy and safety of gemcitabine and cisplatin in patients with unresectable cholangiocarcinoma and gallbladder cancer in daily practice that meet the criteria for the ABC-02 trial in comparison to patients who did not. METHODS: Patients diagnosed with unresectable BTC between 2010 and 2015 with an indication for gemcitabine and cisplatin were included. We divided these patients into three groups: (I) patients who received chemotherapy and met the criteria of the ABC-02 trial, (II) patients who received chemotherapy and did not meet these criteria and (III) patients who had an indication for chemotherapy, but received best supportive care without chemotherapy. Primary outcome was overall survival (OS) and secondary outcome was progression-free survival (PFS). RESULTS: We collected data of 208 patients, of which 138 (66.3%) patients received first line chemotherapy with gemcitabine and cisplatin. Median OS of 69 patients in group I, 63 patients in group II and 65 patients in group III was 9.6 months (95%CI = 6.7-12.5), 9.5 months (95%CI = 7.7-11.3) and 7.6 months (95%CI = 5.0-10.2), respectively. Median PFS was 6.0 months (95%CI = 4.4-7.6) in group I and 5.1 months (95%CI = 3.7-6.5) in group II. Toxicity and number of dose reductions (p = .974) were comparable between the two chemotherapy groups. CONCLUSION: First-line gemcitabine and cisplatin is an effective and safe treatment for patients with unresectable BTC who do not meet the eligibility criteria for the ABC-02 trial. Median OS, PFS and treatment side effects were comparable between the patients who received chemotherapy (group I vs. group II).
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Sistema Biliar/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Neoplasias da Vesícula Biliar/tratamento farmacológico , Cuidados Paliativos/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias do Sistema Biliar/mortalidade , Colangiocarcinoma/mortalidade , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Desoxicitidina/análogos & derivados , Intervalo Livre de Doença , Feminino , Neoplasias da Vesícula Biliar/mortalidade , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , GencitabinaRESUMO
BACKGROUND: Body composition measures may predict outcomes of cancer surgery. Whereas low muscle mass shown on preoperative computed tomography (CT) scans has been associated with worse outcomes after surgery for pancreatic cancer, less consideration has been given to low muscle attenuation, reflecting poor muscle quality. Studies relating muscle mass and muscle attenuation with outcomes for patients with periampullary, nonpancreatic cancer are lacking. METHODS: Skeletal muscle mass and attenuation were assessed in 166 consecutive patients undergoing pancreatoduodenectomy (PD) for periampullary, nonpancreatic cancer at a single center between 2000 and 2012. The skeletal muscle index (SMI) was calculated from cross-sectional muscle area on preoperative CT imaging at the third lumbar vertebra level (L3) and normalized for height. The skeletal muscle attenuation index (MAI) was calculated by measuring the average Hounsfield units of the total muscle area at the L3 level. Overall survival (OS) and the rate of major postoperative complications (Clavien-Dindo ≥3) were extracted from prospectively maintained databases. RESULTS: Low SMI was present in 78.3 % and low MAI in 48.8 % of the patients. The multivariate analysis showed lymph node metastasis [hazard ratio (HR) 1.8; 95 % confidence interval (CI) 1.1-2.9], microscopic radicality (HR 2.0; 95 % CI 1.2-3.4), and low MAI (HR 2.0; 95 % CI 1.2-3.3), but not low SMI to be significantly associated with decreased OS. Low MAI (HR 1.9; 95 % CI 1.0-3.8) was the only independent risk factor for major postoperative complications. CONCLUSION: Skeletal muscle quality, but not muscle mass, predicted survival and major complications after PD for periampullary, nonpancreatic cancer. Preoperative CT-derived body composition measures may stratify patients into risk categories and support shared decision making.
Assuntos
Ampola Hepatopancreática/patologia , Ampola Hepatopancreática/cirurgia , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/cirurgia , Ductos Biliares Extra-Hepáticos/patologia , Ductos Biliares Extra-Hepáticos/cirurgia , Neoplasias Duodenais/patologia , Neoplasias Duodenais/cirurgia , Músculo Esquelético/patologia , Pancreaticoduodenectomia , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/diagnóstico por imagem , Estudos Prospectivos , Taxa de Sobrevida , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Hepatocellular adenoma (HCA) larger than 5 cm in diameter is considered an indication for elective surgery, because of the risk of haemorrhage and malignant transformation. Transarterial embolization (TAE) is used to manage bleeding HCA and occasionally to reduce tumour size. TAE might have potential as an elective therapy, but its current role in this context is uncertain. This systematic review provides an overview of clinical outcomes after TAE, in bleeding and non-bleeding HCA. METHODS: Two independent reviewers performed a systematic search of literature in PubMed and Embase. Outcomes were change in tumour size, avoidance of surgery, complications and malignant transformation after TAE in bleeding and non-bleeding HCA. The Critical Appraisal Skills Programme tool for cohort studies was used for quality assessment of included studies. RESULTS: From 320 potential articles, 20 cohort studies and 20 case reports including 851 patients met the inclusion criteria. TAE was performed in 151 of 851 patients (17·7 per cent), involving 196 tumours, of which 95 (48·5 per cent) were non-bleeding. Surgical treatment was avoided in 68 of 151 patients (45·0 per cent). Elective TAE was performed in 49 patients involving 66 HCAs, with 41 of these patients (84 per cent) not requiring surgery. Major complications occurred in eight of 151 patients (5·3 per cent); no death was reported. Among cohort studies, complete tumour disappearance was observed in 10 per cent of patients, and regression in 75 per cent. CONCLUSION: Acute or elective TAE in the management of HCA is safe. In the elective setting, TAE provides a potential alternative to surgery.
Assuntos
Adenoma de Células Hepáticas/terapia , Embolização Terapêutica , Neoplasias Hepáticas/terapia , Adenoma de Células Hepáticas/complicações , Adenoma de Células Hepáticas/patologia , Hemorragia/etiologia , Hemorragia/terapia , Humanos , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/patologia , Complicações Pós-Operatórias , Resultado do TratamentoRESUMO
Pathological confirmation is desired prior to high-risk surgery for suspected perihilar cholangiocarcinoma (PHC), but preoperative tissue diagnosis is limited by poor sensitivity of available techniques. This study aimed to validate whether a tumor-specific enhanced green fluorescent protein (eGFP)-expressing oncolytic virus could be used for cholangiocarcinoma (CC) cell detection. Extrahepatic CC cell lines SK-ChA-1, EGI-1, TFK-1 and control cells (primary human liver cells) were exposed to the oncolytic herpes simplex type 1 virus NV1066 for up to 24 h in adherent culture. The technique was validated for cells in suspension and cultured cells that had been exposed to crude patient bile. Optimal incubation time of the CC cells with NV1066 at a multiplicity of infection of 0.1 was determined at 6-8 h, yielding 15% eGFP-expressing cells, as measured by flow cytometry. Cells were able to survive 2-h crude bile exposure and remained capable of producing eGFP following NV1066 infection. Detection of malignant cells was possible at the highest dilution tested (10 CC cells among 2 × 105 control cells), though hampered by non-target cell autofluorescence. The technique was not applicable to cells in suspension due to insufficient eGFP production. Accordingly, as yet the technique is not suitable for standardized clinical diagnostics in PHC.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Hepatócitos/metabolismo , Herpesvirus Humano 1/metabolismo , Vírus Oncolíticos/metabolismo , Animais , Ácidos e Sais Biliares/farmacologia , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/virologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Colangiocarcinoma/virologia , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Hepatócitos/citologia , Hepatócitos/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Humanos , Vírus Oncolíticos/genética , Vírus Oncolíticos/fisiologia , Células VeroRESUMO
A novel approach was used to select the most suitable antiviral monoclonal antibody (mAb) and elution conditions for immunoaffinity purification of the NS1 protein of Murray Valley encephalitis virus (MVE). Crude NS1 protein was subjected to a variety of chemical conditions produced by common elution buffers, and tested with a panel of NS1-specific mAbs by ELISA to determine which buffers denatured antigenic epitopes. Buffers that caused least structural damage to NS1 epitopes were tested by ELISA for dissociation of NS1-mAb complexes adsorbed to the solid phase. For each mAb analysed, the conditions required to break the mAb-NS1 complex on the solid phase were similar to those required to release antigen bound to mAb-sepharose beads. From these results we selected an appropriate antibody for affinity purification of the non-structural viral protein NS1 on CNBr-activated sepharose columns. Elution at pH 11.5 yielded good recoveries of highly pure and antigenically intact NS1 dimer. The results demonstrate that the appropriate ligand and optimal elution conditions can be rapidly determined generally by immunoassay in microtitre plates.
Assuntos
Capsídeo/isolamento & purificação , Flavivirus/análise , Proteínas do Core Viral/isolamento & purificação , Anticorpos Monoclonais , Capsídeo/imunologia , Cromatografia de Afinidade , Dietilaminas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Concentração de Íons de Hidrogênio , Cloreto de Magnésio , Proteínas do Core Viral/imunologia , Proteínas não Estruturais ViraisRESUMO
The design of oligonucleotides used for hybridisation studies often utilises available sequence information of the type strain of a particular virus. If hybridisation studies, using such oligonucleotides, are carried out with field isolates of the same virus, the problem of base pair mismatches and consequent difficulties in detection may arise. This study examined the effect of base pair mismatches on the hybridisation between membrane-bound Murray Valley encephalitis virus (MVE) RNA derived from various strains and deliberately mismatched oligonucleotide probes. Under conditions of very low stringency, probes containing up to 5 mismatches were able to detect MVE RNA, but not yeast RNA. Under washing conditions of increased stringency, hybridisation could be detected between MVE virus RNA and probes with only 3 to 4 mismatches. However, the extent of this interaction was dependent on the number and type of mismatches and their relative sequence position.
Assuntos
Flavivirus/isolamento & purificação , Sondas de Oligonucleotídeos , RNA Viral/análise , Animais , Sequência de Bases , DNA Viral , Immunoblotting , Dados de Sequência Molecular , Ácidos Nucleicos Heteroduplexes , Sensibilidade e Especificidade , Células VeroRESUMO
A sensitive nested RT-PCR that can be carried out in a single tube is described. The sensitivity of this system was determined, and compared to that of a single round of PCR, and a single round of PCR followed by hybridisation with a radiolabelled oligonucleotide probe. We found that with the one-tube nested RT-PCR we were able to detect 0.1 pfu/ml of Ross River virus. The nested RT-PCR was 100-times more sensitive than a single round of RT-PCR followed by hybridisation, and 10,000-times more sensitive than a single round of RT-PCR alone. This system provides a sensitive detection of Ross River virus, and can be adapted for detection of RNA from any source. The test material is added to a single tube at the outset, and by subsequent addition of two sets of reagents, the entire nested RT-PCR can be carried out in the same tube. This system has maximum sensitivity, minimises risk of contamination, and is amenable to automation.
Assuntos
Reação em Cadeia da Polimerase/métodos , Ross River virus/genética , Ross River virus/isolamento & purificação , Infecções por Alphavirus/diagnóstico , Animais , Sequência de Bases , Primers do DNA/genética , Sondas de DNA/genética , DNA Viral/genética , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/estatística & dados numéricos , Sensibilidade e Especificidade , Ensaio de Placa Viral , Virologia/métodosRESUMO
A convenient method employing a commercially available apparatus for two-dimensional gel electrophoresis of RNase T1 resistant oligonucleotides using ultrathin gels has been developed. The methodology overcomes problems commonly associated with the establishment of good, bubble-free, fusion of the first and second dimension gels. The use of ultrathin gels results in autoradiograms with well resolved oligonucleotide spots.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Flavivirus/isolamento & purificação , RNA Viral/isolamento & purificação , Ribonuclease T1 , Virologia/métodosRESUMO
A sensitive, single tube reverse transcription-polymerase chain reaction (RT-PCR) protocol for the detection of Ross River virus (RRV) is described. All components necessary for both reverse transcription and PCR were combined in a single tube, and reverse transcription and PCR carried out sequentially in a single, non-interrupted thermal cycling program. The antisense oligonucleotide from the two primers selected for use in the PCR also served to prime specifically for the reverse transcription. The 549 bp product was detected by electrophoresis and ethidium bromide staining. The detection limit using this system was 18 fg of purified viral RNA or 1.3 pfu of whole virus. Greater sensitivity cannot reasonably be expected unless a more sensitive method than electrophoresis and ethidium bromide staining is used for PCR product detection, such as nested PCR or hybridisation with labelled probe. This PCR detection system will be adapted for detection of RRV in mosquito populations for virus surveillance programs.
Assuntos
Reação em Cadeia da Polimerase/métodos , Ross River virus/genética , Virologia/métodos , Animais , Sequência de Bases , Culicidae/microbiologia , DNA Viral/genética , Estudos de Avaliação como Assunto , Humanos , Insetos Vetores/microbiologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase/estatística & dados numéricos , RNA Viral/genética , RNA Viral/isolamento & purificação , Ross River virus/isolamento & purificação , Sensibilidade e Especificidade , Ensaio de Placa Viral , Cultura de VírusRESUMO
Mycoplasma bovis is responsible for several production diseases in cattle, including mastitis, arthritis, pneumonia, abortion and infertility. Current methodologies for detecting and identifying M. bovis are time consuming and difficult. Tests which rely on antigen or antibody detection have poor sensitivity and specificity. In this paper associated protocols for the development of a hybridization probe and PCR are described. A genomic library (SauIIIA digested) was prepared from M. bovis DNA (Colindale Reference Strain: NC10131:02) and cloned into pUC19. Colony hybridization, using a probe preparation made from purified M. bovis DNA, was used to identify colonies of interest. M. bovis DNA fragments were retrieved from recombinant plasmids by digestion with EcoRI and HindIII. This DNA was used to prepare randomly primed probes for dot blot hybridization analysis with immobilized DNA from M. bovis (two strains), M. dispar, M. agalactiae, M. bovigenitalium (two strains), M. ovipneumoniae, a Group 7 strain, M. arginini and bacteria belonging to different genera. Four probes were found to hybridize only with M. bovis and M. ovipneumoniae DNA, whereas one probe reacted with genomic DNA from only one of the two M. bovis strains. The level of sensitivity of the dot blot hybridization assay was 200 CFU (colony forming units)/mL. To enhance the sensitivity further, an M. bovis-specific PCR assay was developed. The primers were designed using sequences obtained from the probe DNA which discriminated M. bovis from all other Mycoplasma DNA tested. The minimum amount of target DNA that could be detected by the PCR assay was that isolated from 10-20 CFU/mL. The PCR assay was therefore 10 times more sensitive than dot blot hybridization.
Assuntos
Sondas de DNA , Mycoplasma/isolamento & purificação , Reação em Cadeia da Polimerase , Animais , Bovinos , Biblioteca Genômica , Leite/microbiologia , Mycoplasma/genética , Infecções por Mycoplasma/diagnóstico , Sensibilidade e EspecificidadeRESUMO
A highly sensitive and specific PCR (MB-PCR) was used in preliminary studies to detect M. bovis in milk samples to investigate its association with high somatic cell count (SCC), an indicator of subclinical mastitis and one of the factors in down grading the quality of milk. A total of 186 and 167 herds were tested with 43% and 62% of herds positive for M. bovis in Victoria and North Queensland, respectively. The quarter milks from 52 cows with persistently high SCC were tested by MB-PCR and culture to investigate the association of M. bovis with major mastitis pathogens (MMP). M. Bovis was detected in 77% of cows of which 19% alone had M. bovis without any other bacteria, 17% had M. bovis in combination with major mastitis pathogens and 40% had M. bovis in combination with non-major mastitis pathogens. We believe that M. bovis is widespread in dairy cattle and has the potential to produce disease alone or to predispose the udder to disease caused by major mastitis and environmental pathogens. These studies have revealed a hitherto unrecognised high prevalence of M. bovis in dairy cattle in North Queensland and Victoria in Australia. These initial studies also give a clear association between M. bovis and elevated somatic cell counts.
Assuntos
Mastite Bovina/epidemiologia , Leite/microbiologia , Infecções por Mycoplasma/veterinária , Mycoplasma/isolamento & purificação , Animais , Bovinos , Contagem de Células/veterinária , DNA Bacteriano/análise , Feminino , Mastite Bovina/diagnóstico , Leite/química , Mycoplasma/genética , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/epidemiologia , Projetos Piloto , Reação em Cadeia da Polimerase/veterinária , Prevalência , Queensland/epidemiologia , Vitória/epidemiologiaRESUMO
BACKGROUND AND AIMS: In the era of expanding costs of healthcare, this study was conducted to perform a cost analysis of introducing a laparoscopic liver surgery programme for left sided liver lesions. MATERIALS AND METHODS: Consecutive patients treated by laparoscopic liver resections of left lateral segments were included. Controls were a group of 14 patients undergoing open resection for similar pathology. Primary outcomes were costs. Secondary outcomes were complications, conversions, blood loss, length of operation, and length of hospital stay. RESULTS: The laparoscopic approach for hepatic left lateral resection (bisegmentectomy 2 and 3) was performed in fourteen patients (group I, median age 54 [range 26-82] years). In the open group, fourteen patients from a prospectively collected database with the same type of resection were selected (group II, median age 64 [range 29-76] years). Costs of theatre usage in the laparoscopic group were significantly lower (p=0.031). No significant differences in costs of disposable instruments, ward stay and total costs were observed between the two groups. There were three complications in the laparoscopic group compared with two complications in the open group. In the laparoscopic group there were 2 conversions (14%). Median blood loss was significantly lower in the laparoscopic group (50 mls [range 0-750], (p=0.001) versus the open group (500 mls [range 150-750]). Furthermore, operation time was also significantly lower in the laparoscopic group (116 [range 85-261] minutes) versus the open group (165 [range 96-217] minutes, p=0.016). Median length of stay was 6 [range 4-11] days in group I versus 6 [range 5-13] days in group II (p=0.508). CONCLUSION: Costs of laparoscopic liver resections proved to be equivalent to open surgery. Furthermore, implementation of a laparoscopic liver resection programme seems feasible and safe with reduced blood loss and operation time and comparable morbidity and length of stay.
Assuntos
Custos Hospitalares , Hospitais Universitários/economia , Laparoscopia/economia , Neoplasias Hepáticas/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Perda Sanguínea Cirúrgica , Efeitos Psicossociais da Doença , Custos e Análise de Custo , Feminino , Humanos , Tempo de Internação , Neoplasias Hepáticas/economia , Masculino , Pessoa de Meia-Idade , Países BaixosRESUMO
The 5' non-coding region of the genomes of 11 isolates of Murray Valley encephalitis virus from Australia and Papua New Guinea were examined by primer extension sequencing. Although the 5' non-coding region of all isolates was found to be highly conserved, three isolates were significantly different in that they contained extra uridine residues. Two of these isolates from Papua New Guinea contained an extra uridine residue, nominally positioned after nucleotide 54, which was absent from all but one of the Australian isolates tested. This isolate (OR 156) contained a further uridine residue at the same site. These results provide further support for earlier observations on the genetic relationships between these isolates, in particular that OR 156 is more closely related to the Papua New Guinea strains than to the Australian strains.
Assuntos
Flavivirus/genética , RNA Viral/genética , Animais , Austrália , Sequência de Bases , Humanos , Dados de Sequência Molecular , Papua Nova Guiné , Homologia de Sequência do Ácido Nucleico , Células VeroRESUMO
The genomes of 21 isolates of Murray Valley encephalitis virus (MVE) from Australia and Papua New Guinea were characterized and compared using RNase T1 oligonucleotide fingerprinting. Most Australian isolates grouped in clusters that were linked with a similarity coefficient of greater than 75%, indicating substantial homogeneity. Two isolates grouped as a cluster that linked with other isolates at a level of 67%. These two isolates, one from the north and one from the south-east of Australia were very similar and could demonstrate the movement of MVE between these areas. This notion is substantiated by genetic homogeneity of isolates from the Kimberley region and from south-eastern Australia. One Australian isolate (OR 156) and the Papua New Guinea isolate (MK 6684) were substantially different from each other as well as from the other isolates. No evidence was found for a poly(A) tract in the genome of MVE.
Assuntos
Flavivirus/genética , Variação Genética , RNA Viral/análise , Animais , Austrália , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Flavivirus/classificação , Humanos , Papua Nova Guiné , Células VeroRESUMO
The genomes of 22 isolates of Kunjin virus (KUN) from Australia were characterized and compared using RNase T1 oligonucleotide fingerprinting. The results show that all isolates belonged to one topotype, the distribution of which covered the entire Australian continent. This finding is similar to that of Murray Valley encephalitis virus, but in contrast to the results reported for some other flaviviruses such as Saint Louis encephalitis virus.
Assuntos
Flavivirus/genética , Austrália , Mapeamento de Nucleotídeos , RNA Viral/genéticaRESUMO
Murray Valley encephalitis (MVE) virus strain OR2 was serially passaged on Vero cells to establish a persistent infection which was maintained for over 300 days. Supernatants from infected cells protected Vero cells from c.p.e. and caused up to a 95% reduction of wild-type virus yield. These protective and interfering effects suggest that defective interfering (DI) particles are responsible for the establishment and maintenance of the MVE virus persistent infection. The persistently infected cell supernatant preparations shared several features with DI particle preparations from other viral systems, such as their amplification to detectable levels after two to four passages of virus. However, results from this study suggest that DI particles of MVE virus differ from other studied systems in that they are able to affect only moderately the yield of infectious wild-type virus. The genetic drift of the parental virus during the course of a long term persistent infection in vitro appears to be minimal.
Assuntos
Vírus Defeituosos/fisiologia , Flavivirus/fisiologia , Animais , Efeito Citopatogênico Viral , Vírus Defeituosos/genética , Flavivirus/genética , Imunofluorescência , Frequência do Gene , RNA Viral/análise , Inoculações Seriadas , Células VeroRESUMO
Detection of viral RNA by polymerase chain reaction (PCR) requires the prior reverse transcription of the viral RNA. In order to minimise the number of manual manipulations required for processing large numbers of samples, we attempted to design a system whereby all the reagents required for both reverse transcription and amplification can be added to one tube and a single, non-interrupted thermal cycling program performed. Whilst attempting to set up such a one-tube system with Taq polymerase (Taq; Biotech International) and avian myoblastosis virus (AMV) reverse transcriptase (RT), we noticed a substantial decrease in the sensitivity of detection of viral RNA. Investigation of this phenomenon has revealed direct interference of RT with Taq polymerase. Evidence supporting this conclusion includes the following observations: (1) increasing the ratio of Taq to RT improves sensitivity; (2) adding non-homologous RNA improves sensitivity; (3) RT that has been heat inactivated prior to Taq addition does not exert this effect; (4) the effect is not sequence restricted; (5) the Mg2+ ions are not sequestered by RT. In addition, the effect is not limited to AMV RT, Moloney murine leukaemia virus RT also affects Taq activity.
Assuntos
Vírus da Mieloblastose Aviária/enzimologia , Vírus da Leucemia Murina de Moloney/enzimologia , Inibidores da Síntese de Ácido Nucleico , DNA Polimerase Dirigida por RNA/metabolismo , Sequência de Bases , Amplificação de Genes , Magnésio/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Viral/metabolismo , Sensibilidade e Especificidade , Taq Polimerase , TemperaturaRESUMO
BACKGROUND: Ross River virus (RRV) is a mosquito borne alphavirus that has been found in Australia, Papua New Guinea and the Pacific Islands. It is aetiological agent of epidemic polyarthritis, a debilitating illness whose symptoms are arthritis, arthralgia, lethargy, rash and fever which may persist for weeks or months. Diagnosis is made on a serological basis, but in many cases is presumptive rather than definite. OBJECTIVES: To apply the polymerase chain reaction (PCR) to detection of RRV in human sera to assess its suitability for application in disease diagnosis. STUDY DESIGN: Sensitivity of the nested RT-PCR assay was determined by detection of virus of known titre diluted in uninfected serum. Clinical serum samples from patients serologically diagnosed of having RRV infection were tested by nested RT-PCR to assess its diagnostic value. RESULTS: Sensitivity of the nested RT-PCR assay was determined to be detection of 0.01 PFU of virus stock in 100 mul serum. Clinical samples tested showed that 10 of 26 (38%) serum samples with low or negative (non-diagnostic) virus-specific antibody titres were PCR-positive, whereas all 22 specimens with high antibody titres were PCR-negative. PCR positivity was unaffected by repeated freezing and thawing of samples. CONCLUSIONS: While PCR cannot replace serology as a means of RRV diagnosis, it may be useful in conjunction with serological testing, particularly for forming definitive diagnoses in those samples with low (inconclusive) antibody titres. It is faster and more sensitive than virus isolation by tissue culture, and could also prove useful in investigations of disease pathogenesis.
RESUMO
Polybrene was shown to enhance the adsorption of simian sarcoma virus-simian sarcoma associated virus complex (SSV-1/SSAV-1) and baboon endogenous virus (BaEV) onto cells in culture. A 16- to 18-fold increased adsorption of both viruses occurred at 8 micrograms/ml polybrene within one hour after infection. The polybrene mediated adsorption was found to be inhibited by the addition of tri-sodium citrate to the culture medium, suggesting the involvement of electrostatic forces. This contention was further supported by the demonstration of temperature independence of the polybrene mediated interaction.