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Abstract Achromobacter spp. are increasingly recognized as emerging pathogens in immunocompromised patients or suffering cystic fibrosis, but unusual in immunocompetent hosts or individuals that underwent surgery. In this study we describe two simultaneous events attributable to two different Achromobacter spp. contaminated sources. One event was related to an episode of pseudo-bacteremia due to sodium citrate blood collection tubes contaminated with Achromobacter insuavis and the other to Achromobacter genogroup 20 infection and colonization caused by an intrinsically contaminated chlorhexidine soap solution. Both threatened the appropriate use of antimicrobials. Molecular approaches were critical to achieving the accurate species identification and to assess the clonal relationship, strengthening the need for dedicated, multidisciplinary and collaborative work of microbiologists, specialists in infectious diseases, epidemiologists and nurses in the control of infections to clarify these epidemiological situations.
Resumen Achromobacter spp. son reconocidas con mayor frecuencia como patógenos emergentes en pacientes con fibrosis quística e inmunodeprimidos, pero son inusuales en hospedadores inmunocompetentes o quirúrgicos. En este estudio describimos 2 eventos simultáneos atribuibles a 2 fuentes contaminadas con Achromobacter spp. Uno correspondió a un episodio de seudobacteriemia por tubos de citrato de sodio contaminados con Achromobacter insuavis y el otro a infecciones y colonizaciones debidas al uso de solución jabonosa de clorhexidina intrínsecamente contaminada con Achromobacter genogrupo 20. Ambos episodios pusieron en peligro el uso apropiado de antimicrobianos. Los enfoques moleculares fueron fundamentales para lograr la identificación precisa de las especies y evaluar la relación clonal de los aislamientos, lo que refuerza la necesidad del trabajo perseverante y multidisciplinario de microbiólogos, especialistas en enfermedades infecciosas, epidemiólogos y enfermeras en el control de infecciones para el esclarecimiento de estas situaciones epidemiológicas.
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ABSTRACT The aim of this study was to perform the molecular characterization of conserved and variable regions of feline calicivirus capsid genome in order to investigate the molecular diversity of variants in Brazilian cat population. Twenty-six conjunctival samples from cats living in five public short-term animal shelters and three multicat life-long households were analyzed. Fifteen cats had conjunctivitis, three had oral ulceration, eight had respiratory signs (cough, sneeze and nasal discharge) and nine were asymptomatic. Feline calicivirus were isolated in CRFK cells and characterized by reverse transcription PCR target to both conserved and variable regions of open reading frame 2. The amplicons obtained were sequenced. A phylogenetic analysis along with most of the prototypes available in GenBank database and an amino acid analysis were performed. Phylogenetic analysis based on both conserved and variable region revealed two clusters with an aLTR value of 1.00 and 0.98 respectively and the variants from this study belong to feline calicivirus genogroup I. No association between geographical distribution and/or clinical signs and clustering in phylogenetic tree was observed. The variants circulating in public short-term animal shelter demonstrated a high variability because of the relatively rapid turnover of carrier cats constantly introduced of multiple viruses into this location over time.
Subject(s)
Animals , Cats , Cat Diseases/virology , Calicivirus, Feline/isolation & purification , Calicivirus, Feline/genetics , Caliciviridae Infections/veterinary , Pets/virology , Phylogeny , Brazil , Open Reading Frames , Genome, Viral , Calicivirus, Feline/classification , Caliciviridae Infections/virology , Capsid Proteins/geneticsABSTRACT
Human norovirus are major causative agent of nonbacterial acute gastroenteritis. In general, genogroup (G) II.4 is the most prominent major genotype that circulate in human population and the environment. However, a shift in genotypic trends was observed in Korea in December 2014. In this study, we investigated the trend of norovirus genotype in detail using the database of Acute Diarrhea Laboratory Surveillance (K-EnterNet) in Korea. GII.17 has since become a major contributor to outbreaks of norovirus-related infections and sporadic cases in Korea, although the reason for this shift remain unknown.
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Humans , Diarrhea , Disease Outbreaks , Gastroenteritis , Genotype , Korea , NorovirusABSTRACT
Objective@#To analyze the prevalence, genetic structure and evolutionary characteristics of GⅡ.17 norovirus isolated from the fecal samples of rhesus monkeys in Longhu Mountain of Guangxi Zhuang Autonomous Region.@*Methods@#A total of 400 stool specimens were collected from wild rhesus monkeys from March to August of 2015. The GⅡ.17 norovirus named as GX213 was identified in fecal samples by high-throughput sequencing technology. Reverse transcription-polymerase chain reaction was used to confirm and screen GX213, as well as amplify its complete gene sequence. Then the sequence and phylogenetic analysis of three ORFs of GX213 were constructed by software MEGA 6.0.@*Results@#Two out of 400 fecal samples were positive. The full-length genome of GX213 was 7 565 bp (containing PloyA tail), which was composed of three open reading frames (ORFs): ORF1(10-5112 nt), ORF2(5093-6715 nt)and ORF3(6715-7494 nt), with 20 bp overlapping between ORF1 and ORF2, and 1 bp overlapping between ORF2 and ORF3.Analysis of the complete sequence of GX213 showed that it shared the highest homology with the strain of human GⅡ.17 norovirus CUHK-NS-613 (GenBank ID: KU561248) (99.5% identity), and ORF1 and ORF3 also shared the highest homology with the strain CUHK-NS-613 [99.5% and 99.4% in nucleotide (nt); 99.5% and 99.2% in amino acid (aa), respectively], which was the main cause of human norovirus outbreaks in some regions of Asia from 2014 to 2015. ORF2 sequence analysis showed that it displayed the highest identity (99.4% in nt and 99.8% in aa) to the strain CUHK-NS-491 (GenBank ID: KP698928), only one aa mutation aa245P→S(P1.1 region) was observed in the GX213 VP1 protein. Furthermore, the phylogenetic analysis showed that GX213 was more related to CUHK-NS-613 and CUHK-NS-491 than the strain KM1509 (GenBank ID: KX356908) of GⅡ.17 norovirus recently identified in rhesus monkeys.@*Conclusions@#GX213 belongs to the human GⅡ.17 norovirus variant causing the norovirus outbreaks from 2014 to 2015. Our research suggests that GⅡ.17 norovirus can infect not only humans but also rhesus monkeys.
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Objective To investigate the etiology spectrum of hand , foot and mouth disease ( HFMD) and to analysis the molecular characteristics of three predominant human enterovirus stains in Qingdao in 2013.Methods The total enterovirus (EV) strains and strains of EV71, CVA16 and CVA6 in throat swabs of HFMD cases were detected by using multiplex real time RT-PCR.The full-length of the viral VP1 genes of the EV strains were amplified and sequenced .The sequences were phylogenetically analyzed by using the MEGA5.0 software package .Results A total of 841 patients with mild HFMD and 107 patients with serious HFMD were recruited in this study and 64 .3%of them were positive for EV .The predominant pathogens were EV71 (44.8%), CVA6 (28.2%) and CVA16 (9.5%) in 2013.CVA6 replaced CVA16 as the second most common pathogen for HFMD , accounting for 42.7% of all pathogens in children aged less than 3 years and 22.2%of all pathogens in the serious patients .The proportions of CVA6 in the etiology spectrum showed a downtrend along with the increasing age of the patients (P<0.001).Phylogenetic analy-sis of the complete VP1 gene sequences showed that all of the EV 71 strains identified in this study belonged to the subgenotype C4 (evolutionary branch C4a) and all of the CVA16 strains belonged to the subgenotype B1 (evolutionary branches B1a and B1b).There were 6 genogroups (A to F) regarding to the VP1 gene of CVA6 and all of the CVA6 strains identified in this study belonged to genogroups A and D .Among the CVA6 strains isolated in Qingdao in 2013, 83.9% belonged to genogroup A, while the rest 16.1% belonged to genogroup D.66.7%of the CVA6 strains isolated in 2012 belonged to genogroup A, while the rest 33.3%belonged to genogroup D .All of the CVA6 strains isolated from year 2008 to 2011 in Qingdao belonged to genogroup D.Conclusion EV71, CVA6 and CVA16 were the prevalent pathogens responsible for the de-velopment of HFMD in Qingdao in 2013.The proportions of CVA6 strains in the etiology spectrum showed a downtrend with the increasing age in children .C4a was the major subtype of EV71 strains circulating in Qingdao in 2013, while B1a and B1b were the major subtypes of CVA16 strains.The pattern of endemic cir-culation of CVA6 strains showed a trend of changing from genogroup D to A from year 2008 to 2013 .
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Objective To study the epidemic and genetic characteristics of coxsackievirus A6 ( CVA6) strains isolated in Guangdong province.Methods Enterovirus strains positive for neither entero-virus A71 ( EV71) nor CVA16 were isolated from Guangdong province during 2008 to 2013 to screen CVA6 isolates by real-time PCR.The entire sequences of viral genes encoding VP1 of CVA6 positive samples were amplified and sequenced.The phylogenetic analysis was performed to analyze the full-length gene sequences encoding VP1 of CVA6 isolates and sequences downloaded from GenBank by using DNAStar6.0 and MEGA5.2 software packages.Results CVA6 strains accounted for 61.4%of the 1672 non-EV71 and non-CVA16 enterovirus strains isolated in Guangdong province during year 2008 to 2013.The positive rates were respectively 10.5%(4/38), 66.7%(34/51), 36.2% (81/224), 63.0% (182/289), 62.3% (325/522) and 73.0%(400/548) from 2008 to 2013 and the differences among different years were significant (χ2=133.79, P<0.01).The CVA6 isolates could be classified into four clusters in the phylogenetic tree, designated A, B, C and D (including D1, D2 and D3 subgenogroups) genogroups.The four clusters shared nucleotide diversity ranging from 15.5% to 23.1%.The CVA6 strains isolated in Guangdong province shared 88.7%-100.0% homologies in nucleotide and 95.7%-100.0% in amino acid.Subtype D2 strains circulated during 2008 to 2012 and subtype D3 strains circulated during 2009 to 2013.Conclusion CVA6 strains were the predominant enterovirus strains among non-EV71 and non-CVA16 enterovirus strains circula-ted in Guangdong province from year 2008 to 2013.The CVA6 isolates could be classified into A, B, C and D genogroups based on the sequence analysis of VP1 region.Subgroups D2 and D3 isolates were identified and the subgroup D3 isolates were the prevalent strains in Guangdong.
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Purpose: Noroviruses (NoV) are increasingly recognized as an important cause for acute gastroenteritis, worldwide. Reverse transcription polymerase chain reaction (RT-PCR) and sequencing are the methods of choice for the detection of NoVs, but there is currently no consensus about the primers to be used in these assays. Materials and Methods: In this study, five published primer sets were evaluated for the detection of genogroup II (GII) NoVs in India. The primers target different regions of the NoV genome. Three primer sets detect an NoV in a single round RT-PCR platform, while the remaining two primer sets are based on a nested RT-PCR platform. Result: A panel of 100 samples from previous studies on norovirus diarrhoea in children were tested by all five primer sets. Of them, 74 samples were identified as positive for NoV, by at least one primer set. Subsets of positive amplicons were sequenced to check for specificity. Conclusion: The most sensitive primer set was Girish 2002, which detected GII NoV by nested RT-PCR, and was modified from the previously published primers. This study demonstrates that higher detection can be obtained by either using multiple primer sets or using a sensitive nested RT-PCR assay. It also demonstrates the differences in primer sensitivity for detection of Genogroup II (GII) NoVs in India.
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Caliciviridae Infections/diagnosis , Child, Preschool , DNA Primers/genetics , Gastroenteritis/diagnosis , Humans , India , Infant , Molecular Diagnostic Techniques/methods , Norovirus/classification , Norovirus/genetics , Norovirus/isolation & purification , Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity , Virology/methodsABSTRACT
Objective To study the genomic molecular organization and genogroup of human metapneumovirus(hMPV) infected infants in Guangzhou of China. Methods Primers were designed on the basis of the genomic sequence of hMPV 00-1 strain(AF371337) in the GenBank, and amplify hMPV genomeby RT-PCR. The PCR-products were cloned to T vector and sequenced, the genomic nucleotide sequences were analyzed with the programs Clustal W/X, DNASTAR and MEGA4. 1. Results The cloned strainhMPVgz01 genome is 13 327 bp in length, the genome contains eight open reading frames in the order 3-N-P-M-F-M2-SH-G-L-5. The genomic sequences of hMPVgz01 strain are compared with those of hMPV in GenBank, revealed that the homology with hMPV group A ranges between 92%-97%, homology with group B is 81%, and with avian metapneumovirus group C is 71%, the highest homology is with BJ1887 strain of genogroup A2b. The N, F, G genes of hMPVgz01 strain are compared with those corresponding genes of hMPV subgroups A1, A2, B1, B2, revealed that the highest homology is also with genogroup A2b. Conclusion The complete nucleotide sequence of hMPVgz01 strain isolated from Guangzhou in China is 13 327 bp in length, GenBank accession No. is GQ153651. Comparison of the genomic sequence and three genes of hMPVgz01 strain with those corresponding sequences of hMPV show the highest homology is with genogroup A2b. Sequence and phylogenetic analysis of the hMPVgz01 strain revegled that this isolate belongs to genogroupA2b.
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Norovirus (NoV), previously called Norwalk-like virus, represents an important group of human pathogens associated with outbreaks of nonbacterial gastroenteritis. Epidemiological surveys of outbreaks have shown that the most important routes of transmission are person-to-person contacts and contaminated food and water, with the virus affecting adults and children. NoV is classified into genogroups, being genogroups GI, GII and GIV found in humans. In view of the high genetic diversity of NoV and the lack of information about this virus in Brazil, the aim of the present study was the molecular characterization of NoV isolated from diarrheic stool samples of patients from São Paulo State. In this study, 204 stool specimens collected during diarrhea outbreaks were analyzed by RT-PCR, and 12 were sequenced for genogroup confirmation. One-step PCR was performed in order to amplify the B region of ORF 1 using the MON 431, 432, 433 and 434 primer pool. From total, 32 (15.7 percent) stool specimens were positive for NoV genogroup GII. Comparison of the sequences of the PCR products to GenBank sequences showed 88.8 percent to 98.8 percent identity, suggesting the presence of genogroup GII in gastroenteritis outbreaks in São Paulo State.
Norovírus (NoV), anteriormente chamado Norwalk-like vírus, constituem um importante grupo de patógeno humano associado a surtos de gastroenterites não bacterianas. Levantamentos epidemiológicos de surtos demonstram que as mais importantes vias de transmissão são contatos pessoa-pessoa, água e alimentos contaminados, afetando adultos e crianças. Os NoV são classificados em genogrupos, sendo os genogrupos GI, GII e GIV encontrados em humanos. A grande diversidade genética dos NoV e a falta de informação sobre esses vírus no Brasil determinaram o tema deste estudo que teve como objetivo a identificação e caracterização molecular de NoV em amostras fecais de pacientes com gastroenterites, provenientes de surtos de diarréia no estado de São Paulo. Neste estudo, 204 amostras de fezes coletadas durante surto de diarréia foram analisadas por RT-PCR, e 12 foram seqüenciadas para confirmar o genogrupo. O método one step PCR foi empregado para a amplificação da região B da ORF1 com o pool de primers MON 431, 432, 433 e 434. Do total, 32 (15,7 por cento) amostras foram positivas para norovírus genogrupo GII. A comparação das seqüências dos produtos de PCR às seqüências do GenBank demonstrou 88,8 por cento a 98,8 por cento de identidade, sugerindo a presença de norovírus genogrupo GII em surtos de gastroenterites no Estado de São Paulo.
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Humans , Disease Outbreaks , Genetic Variation , Gastroenteritis/genetics , In Vitro Techniques , Norovirus/genetics , Norovirus/isolation & purification , Critical Pathways , Genetic Techniques , Methods , VirulenceABSTRACT
Objective To develop a rapid, specific and sensitive diagnostic method for quantification and typing of genogroup Ⅰ and Ⅱ norovirus in oyster shellfish and stool samples from patients who had eaten them. Methods Specific primers and probe, following large scale norovirus genome consensus analysis were designed and subsequently a TaqMan based Real-time PCR assay to detect both GⅠ and GⅡ were established. Results This method showed high specificity for norovirus nucleic acid detection, and no cross-reaction among norovirus GⅠ and GⅡ. The limit on detection of NV genomes was 102 copies/μl. A total of 90 oysters and 37 stool specimens with diarrhea were tested for norovirus by conventional reverse transcriptional PCR (RT-PCR) assay as well as the TaqMan Real-time PCR, respectively. The norovirus detection rate in oysters by TaqMan PCR was significantly higher than that by conventional RT-PCR, but no differences between the two PCR methods were found when detecting the stool samples. Reliability of the Real-time PCR for norovirus detection was further confirmed by DNA sequencing of the positive samples.Conclusion This TaqMan Real-time PCR assay was proved to be a useful method for quantification and typing for norovirus in routine monitoring of both oyster shellfish and clinical samples.This method is recommended to be an effective diagnostic method for outbreak-associated gastroenteritis due to norovirus.
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BACKGROUND: TT virus (TTV) infection is highly prevalent in general population and patients with hepatitis B virus (HBV) infection. The aim of the present study was to determine the distribution of the genotypes and genogroups of TTV in healthy and HBV-infected individuals in Korea. METHODS: Distribution of TTV genotypes and genogroups was investigated in the serum samples of 69 healthy and 59 HBV-infected individuals. PCR products of N22 region were genotyped by sequence analysis. TTV genogroups were determined by 5 different genogroup-specific PCR assays. RESULTS: Among the 20 sequenced isolates, 9 (45%) were genotype 2, 8 (40%) were genotype 1, 2 (10%) were genotype 3, and 1 (5%) was genotype 4. TTV genogroup 4 was found most frequently (52/128), followed by genogroup 3 (42/128), genogroup 1 (35/128), genogroup 5 (32/128), and genogroup 2 (1/128). Mixed infections with different genogroups were frequent. CONCLUSIONS: TTV genotype 2 and 1 are predominant genotypes. TTV genotype 3 was detected for the first time in Korea. TTV genogroups 4 and 3 were predominant genogroups. No significant difference was observed in the distribution of TTV genogroups between healthy and HBV-infected individuals.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Amino Acid Sequence , DNA Virus Infections/diagnosis , Genotype , Hepatitis B/complications , Korea , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction/methods , Torque teno virus/classificationABSTRACT
BACKGROUND: Small round structured virus (SRSV) is one of the common etiologic agents of viral gastroenteritis in childhood. However, SRSV has not easily been detected in many diarrheal outbreaks. Therefore, we experienced simultaneous outbreak of SRSV enteritis in two elementary schools, therefore, we analyzed the result of the surveillance. METHODS: We interviewed the pupil of two schools and questioned their demographic data, presence of gastrointestinal symptoms, contact with symptomatic persons within the last two weeks, and dietary history within the last one week in school. The specimens of the symptomatic pupil were collected for identification of bacteria (Shigella spp, Salmonella spp, E.coli O157 : H7) and viruses (Norwalk agent, SRSV, adenovirus, and astrovirus). RESULTS: The number of symptomatic patients was 193 (193/2843, 6.8%) and the distribution of symptom onset was unipolar. Frequent symptoms of the patients were abdominal pain (176/193, 91.2%), headache (111/193, 57.5%), vomiting (102/193, 52.8%), diarrhea (83/ 193, 43.0%), febrile sense (79/193, 40.9%), nausea (73/193, 37.8%), chilling (49/193, 25.4%), and tenesmus (8/193, 4.1%). We identified SRSV in 9 cases by PCR method, and analyzed the RNA polymerase gene by DNA sequencing; 2 genogroup (Genogroup I, II) and 3 genotype. CONCLUSION: We confirmed SRSV enteritis with different genotypes in two concurrent outbreaks. DNA sequencing and comparison of genotype among the isolated SRSV in near future are be necessary
Subject(s)
Humans , Abdominal Pain , Adenoviridae , Bacteria , Diarrhea , Disease Outbreaks , DNA , DNA-Directed RNA Polymerases , Enteritis , Gastroenteritis , Genotype , Headache , Nausea , Polymerase Chain Reaction , Pupil , Salmonella , Sequence Analysis, DNA , VomitingABSTRACT
BACKGROUND: Small round structured virus (SRSV) is one of the common etiologic agents of viral gastroenteritis in childhood. However, SRSV has not easily been detected in many diarrheal outbreaks. Therefore, we experienced simultaneous outbreak of SRSV enteritis in two elementary schools, therefore, we analyzed the result of the surveillance. METHODS: We interviewed the pupil of two schools and questioned their demographic data, presence of gastrointestinal symptoms, contact with symptomatic persons within the last two weeks, and dietary history within the last one week in school. The specimens of the symptomatic pupil were collected for identification of bacteria (Shigella spp, Salmonella spp, E.coli O157 : H7) and viruses (Norwalk agent, SRSV, adenovirus, and astrovirus). RESULTS: The number of symptomatic patients was 193 (193/2843, 6.8%) and the distribution of symptom onset was unipolar. Frequent symptoms of the patients were abdominal pain (176/193, 91.2%), headache (111/193, 57.5%), vomiting (102/193, 52.8%), diarrhea (83/ 193, 43.0%), febrile sense (79/193, 40.9%), nausea (73/193, 37.8%), chilling (49/193, 25.4%), and tenesmus (8/193, 4.1%). We identified SRSV in 9 cases by PCR method, and analyzed the RNA polymerase gene by DNA sequencing; 2 genogroup (Genogroup I, II) and 3 genotype. CONCLUSION: We confirmed SRSV enteritis with different genotypes in two concurrent outbreaks. DNA sequencing and comparison of genotype among the isolated SRSV in near future are be necessary
Subject(s)
Humans , Abdominal Pain , Adenoviridae , Bacteria , Diarrhea , Disease Outbreaks , DNA , DNA-Directed RNA Polymerases , Enteritis , Gastroenteritis , Genotype , Headache , Nausea , Polymerase Chain Reaction , Pupil , Salmonella , Sequence Analysis, DNA , VomitingABSTRACT
Twenty isolates of Hantavirus were isolated from patients and reserovirs from 1988 to 1994 in Korea. Isolation rate was 1.9% (10/538) in patients, 6.2% (5/81) in Apodemus sp., 2.6% (1/38) in Rattus sp. and 0.6% (4/677) in bats. Reciprocal mean IFA titers ranged from 27.5 to 1,024 at the specimen collection. According to the growth rate and reaching peak titer of infectivity, the isolates were grouped as rapid, intermediate, and slow growing groups. All isolates were confirmed as Hantaan type by the nested RT-PCR on the Gl region of the M segment. Comparison of nucleotide sequence (Nt: 2101 - Nt: 2280) of the G2 region revealed that the sequence homology between Hantaan 76/118 virus and the isolates was more than 90%. Several nucleotide positions of the isolates showed high variation. The variation rate of patientisolates was about one-half when compared with that of rodentisolates. On the basis of phylogenetic analysis Hantaan viruses isolated were divided into two genogroups. These results indicate that Hantaan virus is highly dominant serotype in Korea and the virologic property and genogroup are not correlated.