RESUMO
N-linked glycosylation plays a key role in the efficacy of many therapeutic proteins. One limitation to the bacterial glycoengineering of human N-linked glycans is the difficulty of installing a single N-acetylglucosamine (GlcNAc), the reducing end sugar of many human-type glycans, onto asparagine in a single step (N-GlcNAcylation). Here, we develop an in vitro method for N-GlcNAcylating proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. We use cell-free protein synthesis (CFPS) to test promiscuous PglB variants previously reported in the literature for the ability to produce N-GlcNAc and successfully determine that PglB with an N311V mutation (PglBN311V) exhibits increased GlcNAc transferase activity relative to the wild-type enzyme. We then improve the transfer efficiency by producing CFPS extracts enriched with PglBN311V and further optimize the reaction conditions, achieving a 98.6 ± 0.5% glycosylation efficiency. We anticipate this method will expand the glycoengineering toolbox for therapeutic research and biomanufacturing.
Assuntos
Acetilglucosamina , Campylobacter jejuni , Sistema Livre de Células , Glicoproteínas , Hexosiltransferases , Campylobacter jejuni/enzimologia , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Glicosilação , Glicoproteínas/metabolismo , Glicoproteínas/genética , Glicoproteínas/química , Acetilglucosamina/metabolismo , Acetilglucosamina/química , Hexosiltransferases/metabolismo , Hexosiltransferases/genética , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genéticaRESUMO
Glycan-binding receptors known as lectins represent a class of potential therapeutic targets. Yet, the therapeutic potential of targeting lectins remains largely untapped due in part to limitations in tools for building glycan-based drugs. One group of desirable structures is proteins with noncanonical glycans. Cell-free protein synthesis systems have matured as a promising approach for making glycoproteins that may overcome current limitations and enable new glycoprotein medicines. Yet, this approach has not been applied to the construction of proteins with noncanonical glycans. To address this limitation, we develop a cell-free glycoprotein synthesis platform for building noncanonical glycans and, specifically, clickable azido-sialoglycoproteins (called GlycoCAP). The GlycoCAP platform uses an Escherichia coli-based cell-free protein synthesis system for the site-specific installation of noncanonical glycans onto proteins with a high degree of homogeneity and efficiency. As a model, we construct four noncanonical glycans onto a dust mite allergen (Der p 2): α2,3 C5-azido-sialyllactose, α2,3 C9-azido-sialyllactose, α2,6 C5-azido-sialyllactose, and α2,6 C9-azido-sialyllactose. Through a series of optimizations, we achieve more than 60% sialylation efficiency with a noncanonical azido-sialic acid. We then show that the azide click handle can be conjugated with a model fluorophore using both strain-promoted and copper-catalyzed click chemistry. We anticipate that GlycoCAP will facilitate the development and discovery of glycan-based drugs by granting access to a wider variety of possible noncanonical glycan structures and also provide an approach for functionalizing glycoproteins by click chemistry conjugation.
Assuntos
Glicoproteínas , Sialoglicoproteínas , Glicosilação , Lectinas/metabolismo , Polissacarídeos/metabolismo , Sialoglicoproteínas/metabolismo , Sistema Livre de CélulasRESUMO
Enterotoxigenic Escherichia coli (ETEC) is the primary etiologic agent of traveler's diarrhea and a major cause of diarrheal disease and death worldwide, especially in infants and young children. Despite significant efforts over the past several decades, an affordable vaccine that appreciably decreases mortality and morbidity associated with ETEC infection among children under the age of 5 years remains an unmet aspirational goal. Here, we describe robust, cost-effective biosynthetic routes that leverage glycoengineered strains of non-pathogenic E. coli or their cell-free extracts for producing conjugate vaccine candidates against two of the most prevalent O serogroups of ETEC, O148 and O78. Specifically, we demonstrate site-specific installation of O-antigen polysaccharides (O-PS) corresponding to these serogroups onto licensed carrier proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. The resulting conjugates stimulate strong O-PS-specific humoral responses in mice and elicit IgG antibodies that possess bactericidal activity against the cognate pathogens. We also show that one of the prototype conjugates decorated with serogroup O148 O-PS reduces ETEC colonization in mice, providing evidence of vaccine-induced mucosal protection. We anticipate that our bacterial cell-based and cell-free platforms will enable creation of multivalent formulations with the potential for broad ETEC serogroup protection and increased access through low-cost biomanufacturing.
RESUMO
Cell-free protein synthesis systems that can be lyophilized for long-term, non-refrigerated storage and transportation have the potential to enable decentralized biomanufacturing. However, increased thermostability and decreased reaction cost are necessary for further technology adoption. Here, we identify maltodextrin as an additive to cell-free reactions that can act as both a lyoprotectant to increase thermostability and a low-cost energy substrate. As a model, we apply optimized formulations to produce conjugate vaccines for â¼$0.50 per dose after storage at room temperature (â¼22 °C) or 37 °C for up to 4 weeks, and â¼$1.00 per dose after storage at 50 °C for up to 4 weeks, with costs based on raw materials purchased at the laboratory scale. We show that these conjugate vaccines generate bactericidal antibodies against enterotoxigenic Escherichia coli (ETEC) O78 O-polysaccharide, a pathogen responsible for diarrheal disease, in immunized mice. We anticipate that our low-cost, thermostable cell-free glycoprotein synthesis system will enable new models of medicine biosynthesis and distribution that bypass cold-chain requirements.
Assuntos
Escherichia coli , Camundongos , Animais , Vacinas Conjugadas/metabolismo , Escherichia coli/metabolismo , Composição de MedicamentosRESUMO
PURPOSE: We report the first known case of eye findings associated with a Fas-associated protein with death domain (FADD) gene mutation, an exceedingly rare entity. OBSERVATIONS: A 7-year-old boy was referred for decreased vision and eye examination revealed cystoid macular edema and peripheral retinal ischemia in both eyes and progression to tractional retinal detachment in the right eye. CONCLUSIONS AND IMPORTANCE: This case suggests that baseline and annual ophthalmic screening may be beneficial in individuals with FADD mutations. However, greater documentation of cases may be necessary before deriving a clear interval screening recommendation.
RESUMO
Natural products and secondary metabolites comprise an indispensable resource from living organisms that have transformed areas of medicine, agriculture, and biotechnology. Recent advances in high-throughput DNA sequencing and computational analysis suggest that the vast majority of natural products remain undiscovered. To accelerate the natural product discovery pipeline, cell-free metabolic engineering approaches used to develop robust catalytic networks are being repurposed to access new chemical scaffolds, and new enzymes capable of performing diverse chemistries. Such enzymes could serve as flexible biocatalytic tools to further expand the unique chemical space of natural products and secondary metabolites, and provide a more sustainable route to manufacture these molecules. Herein, we highlight select examples of natural product biosynthesis using cell-free systems and propose how cell-free technologies could facilitate our ability to access and modify these structures to transform synthetic and chemical biology.
Assuntos
Produtos Biológicos/metabolismo , Produtos Biológicos/química , Sequenciamento de Nucleotídeos em Larga Escala , Engenharia Metabólica , Estrutura MolecularRESUMO
PURPOSE: We investigated the value of transcriptome sequencing (RNAseq) in ascertaining the consequence of DNA variants on RNA transcripts to improve the diagnostic rate from exome or genome sequencing for undiagnosed Mendelian diseases spanning a wide spectrum of clinical indications. METHODS: From 234 subjects referred to the Undiagnosed Diseases Network, University of California-Los Angeles clinical site between July 2014 and August 2018, 113 were enrolled for high likelihood of having rare undiagnosed, suspected genetic conditions despite thorough prior clinical evaluation. Exome or genome sequencing and RNAseq were performed, and RNAseq data was integrated with genome sequencing data for DNA variant interpretation genome-wide. RESULTS: The molecular diagnostic rate by exome or genome sequencing was 31%. Integration of RNAseq with genome sequencing resulted in an additional seven cases with clear diagnosis of a known genetic disease. Thus, the overall molecular diagnostic rate was 38%, and 18% of all genetic diagnoses returned required RNAseq to determine variant causality. CONCLUSION: In this rare disease cohort with a wide spectrum of undiagnosed, suspected genetic conditions, RNAseq analysis increased the molecular diagnostic rate above that possible with genome sequencing analysis alone even without availability of the most appropriate tissue type to assess.
Assuntos
Doenças Genéticas Inatas/diagnóstico , Patologia Molecular , Doenças Raras/diagnóstico , Transcriptoma/genética , Exoma/genética , Doenças Genéticas Inatas/genética , Testes Genéticos/normas , Humanos , Mutação/genética , RNA-Seq/normas , Doenças Raras/genética , Análise de Sequência de DNA/normas , Sequenciamento do Exoma/normas , Sequenciamento Completo do Genoma/normasRESUMO
Proteases are fundamental to successful parasitism, including that of the schistosome flatworm parasite, which causes the disease schistosomiasis in 200 million people worldwide. The proteasome is receiving attention as a potential drug target for treatment of a variety of infectious parasitic diseases, but it has been understudied in the schistosome. Adult Schistosoma mansoni were incubated with 1 µM concentrations of the proteasome inhibitors bortezomib, carfilzomib, and MG132. After 24 h, bortezomib and carfilzomib decreased worm motility by more than 85% and endogenous proteasome activity by >75%, and after 72 h, they increased caspase activity by >4.5-fold. The association between the engagement of the proteasome target and the phenotypic and biochemical effects recorded encouraged the chromatographic enrichment of the S. mansoni proteasome (Sm20S). Activity assays with fluorogenic proteasome substrates revealed that Sm20S contains caspase-type (ß1), trypsin-type (ß2), and chymotrypsin-type (ß5) activities. Sm20S was screened with 11 peptide epoxyketone inhibitors derived from the marine natural product carmaphycin B. Analogue 17 was 27.4-fold less cytotoxic to HepG2 cells than carmaphycin B and showed equal potency for the ß5 subunits of Sm20S, human constitutive proteasome, and human immunoproteasome. However, this analogue was 13.2-fold more potent at targeting Sm20S ß2 than it was at targeting the equivalent subunits of the human enzymes. Furthermore, 1 µM 17 decreased both worm motility and endogenous Sm20S activity by more than 90% after 24 h. We provide direct evidence of the proteasome's importance to schistosome viability and identify a lead for which future studies will aim to improve the potency, selectivity, and safety.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Caspases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Células Hep G2 , Humanos , Leupeptinas , Oligopeptídeos/farmacologiaRESUMO
IMPORTANCE: Clinical exome sequencing (CES) is rapidly becoming a common molecular diagnostic test for individuals with rare genetic disorders. OBJECTIVE: To report on initial clinical indications for CES referrals and molecular diagnostic rates for different indications and for different test types. DESIGN, SETTING, AND PARTICIPANTS: Clinical exome sequencing was performed on 814 consecutive patients with undiagnosed, suspected genetic conditions at the University of California, Los Angeles, Clinical Genomics Center between January 2012 and August 2014. Clinical exome sequencing was conducted as trio-CES (both parents and their affected child sequenced simultaneously) to effectively detect de novo and compound heterozygous variants or as proband-CES (only the affected individual sequenced) when parental samples were not available. MAIN OUTCOMES AND MEASURES: Clinical indications for CES requests, molecular diagnostic rates of CES overall and for phenotypic subgroups, and differences in molecular diagnostic rates between trio-CES and proband-CES. RESULTS: Of the 814 cases, the overall molecular diagnosis rate was 26% (213 of 814; 95% CI, 23%-29%). The molecular diagnosis rate for trio-CES was 31% (127 of 410 cases; 95% CI, 27%-36%) and 22% (74 of 338 cases; 95% CI, 18%-27%) for proband-CES. In cases of developmental delay in children (<5 years, n = 138), the molecular diagnosis rate was 41% (45 of 109; 95% CI, 32%-51%) for trio-CES cases and 9% (2 of 23, 95% CI, 1%-28%) for proband-CES cases. The significantly higher diagnostic yield (P value = .002; odds ratio, 7.4 [95% CI, 1.6-33.1]) of trio-CES was due to the identification of de novo and compound heterozygous variants. CONCLUSIONS AND RELEVANCE: In this sample of patients with undiagnosed, suspected genetic conditions, trio-CES was associated with higher molecular diagnostic yield than proband-CES or traditional molecular diagnostic methods. Additional studies designed to validate these findings and to explore the effect of this approach on clinical and economic outcomes are warranted.
Assuntos
Exoma , Doenças Genéticas Inatas/diagnóstico , Técnicas de Diagnóstico Molecular , Doenças Raras/diagnóstico , Adolescente , Adulto , Criança , Pré-Escolar , Deficiências do Desenvolvimento/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Doenças Raras/genética , Análise de Sequência de DNA/métodosRESUMO
BACKGROUND AND METHODS: There are considerable uncertainty and debate regarding all aspects of newborn screen-positive cases of 3-methylcrotonyl-CoA carboxylase deficiency (3-MCCD), including diagnostic criteria, clinical spectrum, morbidity, prognosis, and appropriate management. To address some of these questions, we queried data from the California Newborn Screening Program's Screening Information System (SIS) and available scanned laboratory reports on cases of 3-MCCD reported by 15 state contracted metabolic specialty care centers born between July 2005 and December 2010. We evaluated the completeness and utility of the database as a tool for clinical disease characterization. RESULTS: During the study period, 2,959,108 infants were screened and 71 infants were diagnosed with 3-MCCD for an overall incidence of 1:41,676. The availability of diagnostic biochemical laboratory data varied significantly from subject to subject. Using a new case classification based on biochemical severity, we found that 8 of the cases met our criteria for biochemically severe (category 1), 19 cases met our criteria for biochemically mild (category 2) that we suspect to possibly be hypomorphic variants or heterozygote carriers, and 44 cases could not be classified (category 3) as mild or severe based on the data available in SIS. Documentation of the treatment regimens also varied significantly with 49% receiving dietary modification and 44% receiving carnitine. 15% of cases were documented to have experienced at least one of the following symptoms: lethargy, vomiting, irritability, ketosis, poor feeding, or poor tone. The majority of the subjects were completely developmentally age appropriate at their last assessment. CONCLUSIONS: The results suggest that a significant portion of the 3-MCCD "confirmed" cases have a mild biochemical phenotype. Moreover the majority of cases had insufficient data entered to allow for adequate clinical characterization of the cases. These findings raise the concern that a significant number of individuals receiving treatment for 3-MCCD may not have a clinically significant condition. Additionally, the utility of this data system could be improved if centers provided complete confirmatory test results and more specific documentation of clinical outcomes and health/developmental status. Further studies, including a clinical chart review, are necessary to validate the data and further characterize this cohort.
Assuntos
Carbono-Carbono Ligases/deficiência , Triagem Neonatal , Distúrbios Congênitos do Ciclo da Ureia/genética , Acetonitrilas , California , Carbono-Carbono Ligases/genética , Carnitina , Humanos , Lactente , Recém-Nascido , Distúrbios Congênitos do Ciclo da Ureia/epidemiologia , Distúrbios Congênitos do Ciclo da Ureia/patologiaRESUMO
Branchio-oculo-facial syndrome (BOFS; OMIM#113620) is a rare autosomal dominant craniofacial disorder with variable expression. Major features include cutaneous and ocular abnormalities, characteristic facies, renal, ectodermal, and temporal bone anomalies. Having determined that mutations involving TFAP2A result in BOFS, we studied a total of 30 families (41 affected individuals); 26/30 (87%) fulfilled our cardinal diagnostic criteria. The original family with the 3.2 Mb deletion including the TFAP2A gene remains the only BOFS family without the typical CL/P and the only family with a deletion. We have identified a hotspot region in the highly conserved exons 4 and 5 of TFAP2A that harbors missense mutations in 27/30 (90%) families. Several of these mutations are recurrent. Mosaicism was detected in one family. To date, genetic heterogeneity has not been observed. Although the cardinal criteria for BOFS have been based on the presence of each of the core defects, an affected family member or thymic remnant, we documented TFAP2A mutations in three (10%) probands in our series without a classic cervical cutaneous defect or ectopic thymus. Temporal bone anomalies were identified in 3/5 patients investigated. The occurrence of CL/P, premature graying, coloboma, heterochromia irides, and ectopic thymus, are evidence for BOFS as a neurocristopathy. Intrafamilial clinical variability can be marked. Although there does not appear to be mutation-specific genotype-phenotype correlations at this time, more patients need to be studied. Clinical testing for TFAP2A mutations is now available and will assist geneticists in confirming the typical cases or excluding the diagnosis in atypical cases.
Assuntos
Síndrome Brânquio-Otorrenal/genética , Síndrome Brânquio-Otorrenal/patologia , Cromossomos Humanos Par 6/genética , Fenótipo , Fator de Transcrição AP-2/genética , Sequência de Aminoácidos , Sequência de Bases , Deleção Cromossômica , Genótipo , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Análise de Sequência de DNARESUMO
Mitochondrial DNA (mtDNA) depletion syndromes are autosomal recessive conditions in which the mtDNA copy number is greatly decreased in affected tissues. The encephalomyopathic group of these syndromes comprise mutations in SUCLA2 and SUCLG1 subunits [1]. In this report, we describe a patient with fatal infantile lactic acidosis associated with mutations in the SUCLG1 gene and mtDNA depletion. Histological and enzymatic abnormalities in skeletal muscle support the diagnosis of this recently described mitochondrial disorder. This case is unique in that prenatal imaging suggested the diagnosis and that the confirmatory molecular diagnosis was established at 2 weeks of age. We describe prenatal MRI and neonatal laboratory disturbances that can point the clinician toward consideration of this diagnosis when treating infantile lactic acidosis.
Assuntos
Acidose Láctica/genética , DNA Mitocondrial/genética , Doenças Mitocondriais/genética , Mutação , Succinato-CoA Ligases/genética , Acidose Láctica/diagnóstico , Adulto , Evolução Fatal , Feminino , Homozigoto , Humanos , Recém-Nascido , Imageamento por Ressonância Magnética , Doenças Mitocondriais/diagnóstico , Músculo Esquelético/patologia , Gravidez , Sítios de Splice de RNA/genéticaRESUMO
Norovirus (NV) (formerly called Norwalk-like virus) is the most common etiological agent of acute viral gastroenteritis outbreaks worldwide. Recent reports have shown that two new GII.4 variants caused epidemics in Europe. To investigate if it is also the case in Hong Kong, a molecular epidemiological study was undertaken between January 2002 and June 2005. During this period, there was a substantial increase in acute cases of gastroenteritis caused by NV. Phylogenetic analysis showed that GII.2 and GII.4 are the major circulating genotypes. Two new GII.4 variants (variants C and D) were identified in 2002 and 2004, which quickly became the predominant strains. They were almost identical to the variants causing epidemics in Europe recently. Since geographically distinct areas were involved within a short period of time, it is possible that GII.4 has a particular propensity for causing pandemics.
Assuntos
Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Surtos de Doenças , Gastroenterite/epidemiologia , Gastroenterite/virologia , Norovirus/genética , Sequência de Bases , Variação Genética , Hong Kong/epidemiologia , Humanos , Dados de Sequência Molecular , Norovirus/isolamento & purificação , FilogeniaRESUMO
A major product of mitochondrial and peroxisomal beta-oxidation is acetyl-CoA, which is essential for multiple cellular processes. The relative role of peroxisomal beta-oxidation of long chain fatty acids and the fate of its oxidation products are poorly understood and are the subjects of our research. In this report we describe a study of beta-oxidation of palmitate and stearate using HepG2 cells cultured in the presence of multiple concentrations of [U-(13)C(18)]stearate or [U-(13)C(16)] palmitate. Using mass isotopomer analysis we determined the enrichments of acetyl-CoA used in de novo lipogenesis (cytosolic pool), in the tricarboxylic acid cycle (glutamate pool), and in chain elongation of stearate (peroxisomal pool). Cells treated with 0.1 mm [U-(13)C(18)]stearate had markedly disparate acetyl-CoA enrichments (1.1% cytosolic, 1.1% glutamate, 10.7% peroxisomal) with increased absolute levels of C20:0, C22:0, and C24:0. However, cells treated with 0.1 mm [U-(13)C(16)]palmitate had a lower peroxisomal enrichment (1.8% cytosolic, 1.6% glutamate, and 1.1% peroxisomal). At higher fatty acid concentrations, acetyl-CoA enrichments in these compartments were proportionally increased. Chain shortening and elongation was determined using spectral analysis. Chain shortening of stearate in peroxisomes generates acetyl-CoA, which is subsequently used in the chain elongation of a second stearate molecule to form very long chain fatty acids. Chain elongation of palmitate to stearate appeared to occur in a different compartment. Our results suggest that 1) chain elongation activity is a useful and novel probe for peroxisomal beta-oxidation and 2) chain shortening contributes a substantial fraction of the acetyl-CoA used for fatty acid elongation in HepG2 cells.
Assuntos
Ácidos Graxos/metabolismo , Peroxissomos/metabolismo , Acetilcoenzima A/metabolismo , Isótopos de Carbono , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Humanos , Lipídeos/análise , Lipídeos/biossíntese , Mitocôndrias/metabolismo , Oxirredução , Ácidos Palmíticos/metabolismo , Ácidos Esteáricos/metabolismoRESUMO
Severe acute respiratory syndrome (SARS) is thought to be caused by a novel coronavirus, SARS-associated coronavirus. We studied viral shedding of SARS coronavirus to improve diagnosis and infection control. Reverse-transcriptase PCR was done on 2134 specimens of different types. 355 (45%) specimens of nasopharyngeal aspirates and 150 (28%) of faeces were positive for SARS coronavirus RNA. Positive rates peaked at 6-11 days after onset of illness for nasopharyngeal aspirates (87 of 149 [58%], to 37 of 62 [60%]), and 9-14 days for faeces (15 of 22 [68%], to 26 of 37 [70%]). Overall, peak viral loads were reached at 12-14 days of illness when patients were probably in hospital care, which would explain why hospital workers were prone to infection. Low rate of viral shedding in the first few days of illness meant that early isolation measures would probably be effective.
Assuntos
Coronavirus/isolamento & purificação , Síndrome Respiratória Aguda Grave/virologia , Eliminação de Partículas Virais , Adulto , Idoso , Fezes/virologia , Feminino , Humanos , Transmissão de Doença Infecciosa do Paciente para o Profissional , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , RNA Viral/análise , Sistema Respiratório/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Síndrome Respiratória Aguda Grave/diagnóstico , Síndrome Respiratória Aguda Grave/transmissão , Carga ViralRESUMO
Noroviruses (Norwalk-like viruses (NLV)) are recognised as major causes of acute gastroenteritis worldwide. Numerous studies had been carried out on the molecular epidemiology of norovirus in outbreaks but relatively few on sporadic cases. In this study, the molecular epidemiology of noroviruses in sporadic and outbreak cases of acute gastroenteritis in Hong Kong was examined over a 12-month period from July 2001 to June 2002. Specimens from three groups of patients were used in this study. Nine hundred ninety-five specimens from patients enrolled in the Acute Diarrhoeal Diseases Surveillance Programme of the Department of Health, Hong Kong Government; 735 clinical specimens from hospital patients with acute gastroenteritis, and 122 specimens from 44 norovirus outbreaks. Ninety-two (9.2%) surveillance specimens were positive for norovirus RNA by reverse transcription-polymerase chain reaction (RT-PCR), compared to 123 (16.7%) clinical and 101 (82.8%) outbreak specimens. For the first 6 months of the study period, the predominant strain was the Bristol strain that belongs to genogroup II (GII). In the latter 6 months of the study, genogroup I (GI) and strains belonging to other clusters of GII were seen more commonly. The vast majority of strains belonging to the Bristol virus cluster were closely related to the 95/96-US subset that was associated with pandemic infection from 1995 onwards. This study clearly establishes the importance of norovirus as a cause of sporadic cases of acute gastroenteritis in all age groups in Hong Kong.
Assuntos
Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Surtos de Doenças , Gastroenterite/epidemiologia , Gastroenterite/virologia , Norovirus/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Variação Genética , Hong Kong/epidemiologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Norovirus/classificação , Norovirus/isolamento & purificação , Filogenia , RNA Viral/genética , RNA Viral/isolamento & purificaçãoRESUMO
A novel polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) of the hsp65 gene was used for the routine identification of mycobacteria in a high throughput clinical laboratory. A total of 2036 clinical isolates were tested by PRA in conjunction with other methods. The PRA identification of M. tuberculosis complex was 100% sensitive and specific, and 74.5% of nontuberculous mycobacteria (NTM) were correctly identified. It gave highly consistent results for Mycobacterium avium complex (MAC) species and for most isolates of M. fortuitum, M. chelonae, and M. kansasii. It had proven to be highly robust and stable despite usage on such a large-scale and is thus particularly suitable for use in high throughput laboratories in areas with a high incidence of tuberculosis.