RESUMO
BACKGROUND: Necrotizing enterocolitis (NEC) is the most common and serious gastrointestinal disorder among preterm neonates. We aimed to assess a specific gut microbiota profile associated with NEC. METHODS: Stool samples and clinical data were collected from 4 geographically independent neonatal intensive care units, over a 48-month period. Thirty stool samples from preterm neonates with NEC (n = 15) and controls (n = 15) were analyzed by 16S ribosomal RNA pyrosequencing and culture-based methods. The results led us to develop a specific quantitative polymerase chain reaction (qPCR) assay for Clostridium butyricum, and we tested stool samples from preterm neonates with NEC (n = 93) and controls (n = 270). We sequenced the whole genome of 16 C. butyricum strains, analyzed their phylogenetic relatedness, tested their culture supernatants for cytotoxic activity, and searched for secreted toxins. RESULTS: Clostridium butyricum was specifically associated with NEC using molecular and culture-based methods (15/15 vs 2/15; P < .0001) or qPCR (odds ratio, 45.4 [95% confidence interval, 26.2-78.6]; P < .0001). Culture supernatants of C. butyricum strains from preterm neonates with NEC (n = 14) exhibited significant cytotoxic activity (P = .008), and we identified in all a homologue of the ß-hemolysin toxin gene shared by Brachyspira hyodysenteriae, the etiologic agent of swine dysentery. The corresponding protein was secreted by a NEC-associated C. butyricum strain. CONCLUSIONS: NEC was associated with C. butyricum strains and dysbiosis with an oxidized, acid, and poorly diversified gut microbiota. Our findings highlight the plausible toxigenic mechanism involved in the pathogenesis of NEC.
Assuntos
Clostridium butyricum/genética , Disbiose/complicações , Disbiose/microbiologia , Enterocolite Necrosante/complicações , Enterocolite Necrosante/microbiologia , Sobrevivência Celular , Estudos de Coortes , Disbiose/epidemiologia , Enterocolite Necrosante/epidemiologia , Fezes/microbiologia , França/epidemiologia , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Células JurkatRESUMO
Better understanding of the correlation between high-risk HPV DNA testing, viral load quantitation, and E6/E7 mRNA detection is required. The aim of this study was to assess the relationship between these markers and the severity of cervical lesions. One-hundred and fifty one directed cervical specimens were analysed (normal, cervical intraepithelial neoplasia, and cancer). HPV types 16, 18, 31, 33, and 45 DNA detection and quantititation and E6/E7 mRNA detection were performed. DNA was detected in 87 (57.6%) samples and increased from 0% (normal) to 93.9% (cancer). E6/E7 mRNA was detected in 65 (43%) samples and increased with the severity of the lesions from 0% (normal) to 78.8% (26/33) (cancers) (P < 0.001). HPV DNA and E6/E7 mRNA detection were compared in the 141 samples harbouring HPV16, 18, 31, 33, or 45 infection: 45.4% (64/141) of specimens were DNA-/mRNA-, 46% (65/141) were DNA + /mRNA+ and 8.5% (12/141) were DNA + /mRNA-. The proportion of DNA + /mRNA+ specimens increased with the severity of the lesions (P < 0.001). All normal cervix specimens were DNA-/mRNA-. Among grade 2 cervical intraepithelial neoplasia, prevalence of DNA was higher than that of mRNA: 41.6% (5/12) versus 25% (3/12), whereas it was 79.3% (46/58) versus 62% (36/58) among grade 3 cervical intraepithelial neoplasia. Full concordance was observed in cancers as all the 26 DNA+ specimens were mRNA +. Median overall HPV load was higher in DNA + /mRNA+ than in DNA + /mRNA- specimens (1.41 × 10(6) vs. 9.1 × 10(2) copies per million cells, P < 0.001). Both E6/E7 mRNA detection and concordant DNA + /mRNA+ detection increases with the severity of the lesions and with the HPV DNA load.
Assuntos
Proteínas Oncogênicas Virais/biossíntese , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/patologia , RNA Mensageiro/análise , Índice de Gravidade de Doença , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Estudos Transversais , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , RNA Mensageiro/genética , RNA Viral/análise , RNA Viral/genética , Neoplasias do Colo do Útero/virologiaRESUMO
Here, we sequenced the 5,419,609 bp circular genome of an Enterobacter aerogenes clinical isolate that killed a patient and was resistant to almost all current antibiotics (except gentamicin) commonly used to treat Enterobacterial infections, including colistin. Genomic and phylogenetic analyses explain the discrepancies of this bacterium and show that its core genome originates from another genus, Klebsiella. Atypical characteristics of this bacterium (i.e., motility, presence of ornithine decarboxylase, and lack of urease activity) are attributed to genomic mosaicism, by acquisition of additional genes, such as the complete 60,582 bp flagellar assembly operon acquired "en bloc" from the genus Serratia. The genealogic tree of the 162,202 bp multidrug-resistant conjugative plasmid shows that it is a chimera of transposons and integrative conjugative elements from various bacterial origins, resembling a rhizome. Moreover, we demonstrate biologically that a G53S mutation in the pmrA gene results in colistin resistance. E. aerogenes has a large RNA population comprising 8 rRNA operons and 87 cognate tRNAs that have the ability to translate transferred genes that use different codons, as exemplified by the significantly different codon usage between genes from the core genome and the "mobilome." On the basis of our findings, the evolution of this bacterium to become a "killer bug" with new genomic repertoires was from three criteria that are "opportunity, power, and usage" to indicate a sympatric lifestyle: "opportunity" to meet other bacteria and exchange foreign sequences since this bacteria was similar to sympatric bacteria; "power" to integrate these foreign sequences such as the acquisition of several mobile genetic elements (plasmids, integrative conjugative element, prophages, transposons, flagellar assembly system, etc.) found in his genome; and "usage" to have the ability to translate these sequences including those from rare codons to serve as a translator of foreign languages.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Enterobacter aerogenes/genética , Genoma Bacteriano , Rizoma/genética , Composição de Bases , Códon , Enterobacter aerogenes/classificação , Enterobacter aerogenes/efeitos dos fármacos , Perfilação da Expressão Gênica , Ordem dos Genes , Genes Essenciais , Humanos , Klebsiella/classificação , Klebsiella/genética , Dados de Sequência Molecular , Fenótipo , Plasmídeos/genética , Proteoma , RNA Ribossômico 16SRESUMO
The clinical utility of HPV 16 and 18 viral loads remains debated. The aim of this study was to assess the clinical significance of HPV 16 and 18 viral load and to determine a cut-off for optimal prediction of grade 2 or higher cervical intraepithelial neoplasia among patients referred to colposcopy. A total of 186 cervico-vaginal specimens harboring HPV 16 and/or 18 obtained at the time of colposcopy from patients without previous cervical neoplasia were tested for HPV 16 and 18 detection and quantitation using quantitative duplex real-time PCR method. Grade 2 or higher cervical intraepithelial neoplasia was diagnosed in 87 (46.8%) cases. Only HPV 16 median viral load increased significantly with the lesion grade: 9.1 × 10(4) in normal cervix or grade 1 cervical intraepithelial lesion versus 4.0 × 10(6) copies per million cells in grade 2 or higher cervical intraepithelial lesion (P < 0.001). The highest predictive value for grade 2 or higher cervical intraepithelial lesion was observed with a HPV 16 viral load cut-off of 3.0 × 10(6) copies per million cells (91% specificity, 58.2% sensitivity). Using this cut-off, the highest predictive value of HPV 16 viral load was observed among those referred for previous low-grade abnormal cervical cytology (96.4% specificity, 88% sensitivity). HPV 18 quantitation showed very poor predictive value. Specific attention should be given when performing colposcopic examination of women with an HPV 16 viral load higher than 3.0 × 10(6) copies per million cells, especially among those referred after a low-grade abnormal cytology.
Assuntos
Colposcopia , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/isolamento & purificação , Carga Viral , Adolescente , Adulto , Idoso , DNA Viral/análise , Feminino , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia , Esfregaço Vaginal , Adulto Jovem , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/patologia , Displasia do Colo do Útero/virologiaRESUMO
Management of patients infected with high-risk HPV (hrHPV) despite normal colposcopy following abnormal cytology remains a clinical challenge. The aim of this study was to evaluate if, in that specific population, initial HPV 16 and HPV 18 viral loads are predictive of infection clearance over a 24-month follow-up. A total of 67 women infected with hrHPV having normal colposcopy following equivocal or low-grade cytological abnormalities were recruited and attended regular follow-ups based on repeat colposcopies and HPV testing. HPV16 and HPV18 infection were diagnosed in 36 (53.7%) and 7 (10.4%) cases, respectively. Viral load was quantified using the quantitative duplex real-time PCR method. Although this was not observed for HPV 18, initial HPV 16 viral load was highly associated to HPV 16 infection outcome (receiver operating characteristic curve analysis, area under curve: 0.90). Thus, women who had cleared their HPV 16 infection had significantly lower median initial HPV 16 viral load than those with persistent HPV 16 infection: 1.5 × 10(3) copies per million cells (CPMC) versus 3.8 × 10(6) CPMC, respectively (P = 0.006). The best prediction of HPV 16 clearance was obtained with an initial HPV 16 viral load of <7.5 × 10(4) CPMC: 86.7% specificity and 85.7% sensitivity. Finally, six patients were diagnosed with grade 2 or 3 cervical or vaginal intraepithelial neoplasia. Although all had a persistent hrHPV infection, neither HPV 16 nor 18 viral loads were found to be predictive of the risk of cervical or vaginal intraepithelial neoplasia. HPV16 viral load quantitation could represent a clinically useful marker in that very specific population.
Assuntos
Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/isolamento & purificação , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Displasia do Colo do Útero/epidemiologia , Carga Viral , Adulto , Idoso , Colposcopia , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/complicações , Prognóstico , Medição de Risco , Sensibilidade e Especificidade , Resultado do Tratamento , Adulto Jovem , Displasia do Colo do Útero/patologiaRESUMO
The persistence of high-risk HPV (HR-HPV) infection is necessary for the development of cervical intraepithelial neoplasia. The aim of this study was to evaluate if HR-HPV typing and HPV16, 18, 31, and 33 quantitation are predictive for type-specific infection persistence and/or the development of CIN in women under 30 with normal cervical cytology. Young women (under 30) attending a family planning clinic who were HPV positive with normal cervical cytology were included. HPV genotyping was assessed by MY09/MY11 PCR, sequencing, phylogenetic analysis, and cloning when necessary. HR-HPV viral load was quantified using duplex real-time PCR. Study patients were offered for a second smear and HR-HPV detection and quantitation after 12 months. HR-HPV was identified in 43 (21.9%) of the 199 included women. Of these, 39 patients had a second cervical sample taken within a mean interval of 11.7 months (8.8-18.3 months). The mean HR-HPV 16, 18, 31, and 33 initial viral load was 1.9 × 10(6) copies/million cells. The level of viral load did not reveal any significant association with type-specific HR-HPV persistence or the subsequent development of cervical intraepithelial neoplasia. Only HPV16 infection was significantly more likely to persist (91.7% vs. 33.1%, P=0.001) and to develop CIN (33.3% vs. 3.7%, P=0.025). In women under 30 with normal cytology, HR-HPV viral load is common and is not predictive of HPV persistence or the development of cervical intraepithelial neoplasia. HPV16 positive women are significantly more likely to have persistent infection and to develop cervical intraepithelial neoplasia.
Assuntos
Colo do Útero/fisiologia , Colo do Útero/virologia , Papillomaviridae/classificação , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/virologia , Carga Viral , Adolescente , Adulto , Análise por Conglomerados , DNA Viral/química , DNA Viral/genética , DNA Viral/isolamento & purificação , Feminino , Genótipo , Humanos , Papillomaviridae/genética , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo Genético , Prevalência , Análise de Sequência de DNA , Adulto JovemRESUMO
Self-sampling using vaginal swabs could be a valuable alternative to screen for cervical cancer for women who do not attend regular cytological screening. The aim of this study was to determine the prevalence of high and low-risk HPV types and of HPV type 16 and 18 DNA load in self-collected vaginal swabs from 35- to 69-year-old Southern French women of low socioeconomic level or migrant populations who do not attend regular cervical screening. A good concordance (93.1%) was found between cervical brush and vaginal swabs in 29 samples. Self-collected vaginal swabs were examined from 120 women. HPV infection was found in 28 women (23.3%; median age 48 years), 17 (14.1%) of whom harbored high-risk HPV types. HPV type 16 was the high risk type found most frequently, followed by types 53, 31, 18, 58, and 66. The low-risk type detected most frequently was HPV type 6, followed by types 61, 70, and 81. The mean HPV 16 and 18 load was 6.3 log(10) copies/10(6) cells and 2.4 log(10) copies/10(6) cells, respectively. These results suggest that vaginal self-swabs can be a reliable tool for cervical cancer screening in non-attending and inadequately screened elderly women.
Assuntos
Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/isolamento & purificação , Programas de Rastreamento/métodos , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/epidemiologia , Autoadministração , Vagina/virologia , Adulto , Idoso , Feminino , França/epidemiologia , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/virologia , Prevalência , Carga ViralRESUMO
The present study aimed to assess whether hepatitis E virus (HEV) is present in domestic pigs in Southern France, and to determine the relationship between HEV sequences detected from pigs and from humans. Two hundred fifteen sera, 207 stools, and 107 bile samples were collected from 3- or 6-month-old pigs from different regions of Southern France. Pig IgG anti-HEV antibodies testing was performed using a commercial ELISA kit with minor modifications. Pig HEV RNA was tested by real-time PCR and sequencing assays using "in-house" protocols. Forty percent of pigs were HEV-seropositive. Sixty-five percent of 3-month-old pigs and none of 6-month-old pigs were HEV RNA-positive. HEV RNA was significantly more frequently detected from stools than from sera (65% vs. 22%; P < 0.001). Phylogenetic analysis showed that pig HEV sequences belonged to genotype 3 and formed two clusters of genotype 3f and 3e. Nucleotide homology between pig HEV sequences of each cluster was high (>97%), and clusters were correlated with the geographical origin of pigs and with their repartition into pens and buildings in the pig farm. Based on analysis of 331 nucleotides, pig HEV sequences were close genetically to HEV sequences found from humans or pigs in Europe, and one showed complete nucleotide identity with an HEV sequence obtained in France from a human. The present data indicate that 3-month-old pigs from Southern France might represent a potential source of HEV transmission to humans, and stress the potential of HEV to cause epizootic infections in population of farm pigs.
Assuntos
Vírus da Hepatite E/isolamento & purificação , Hepatite E/veterinária , Doenças dos Suínos/virologia , Animais , Bile/virologia , Análise por Conglomerados , Ensaio de Imunoadsorção Enzimática/métodos , Fezes/virologia , França , Genótipo , Geografia , Anticorpos Anti-Hepatite/sangue , Hepatite E/transmissão , Humanos , Imunoglobulina G/sangue , Epidemiologia Molecular , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Análise de Sequência de DNA , Homologia de Sequência , Soro/virologia , SuínosRESUMO
BACKGROUND: Bacterial vaginosis (BV) is a poorly detected public health problem that is associated with preterm delivery and for which no reliable diagnostic tool exists. METHODS: Molecular analysis of 231 vaginal samples, classified by Gram stain-based Nugent score, was used to propose molecular criteria for BV; these criteria were prospectively applied to 56 new samples. A quantitative molecular tool targeting 8 BV-related microorganisms and a human gene was developed using a specific real-time polymerase chain reaction assay and serial dilutions of a plasmid suspension. The targeted microorganisms were Gardnerella vaginalis, Lactobacillus species, Mobiluncus curtisii, Mobiluncus mulieris, and Candida albicans (which can be identified by Gram staining), as well as Atopobium vaginae, Mycoplasma hominis, and Ureaplasma urealyticum (which cannot be detected by Gram staining). RESULTS: With use of the Nugent score, 167 samples were classified as normal, 20 were classified as BV, and 44 were classified as intermediate. Except for U. urealyticum, M. mulieris, and Lactobacillus species, DNA of the tested bacteria was detected more frequently in samples demonstrating BV, but the predictive value of such detection was low. The molecular quantification of A. vaginae (DNA level, > or = 10(8) copies/mL) and G. vaginalis (DNA level, > or = 10(9) copies/mL) had the highest predictive value for the diagnosis of BV, with excellent sensitivity (95%), specificity (99%), and positive (95%) and negative (99%) predictive values; 25 (57%) of the samples demonstrating intermediate flora had a BV profile. When applied prospectively, our molecular criteria had total positive and negative predictive values of 96% and 99%, respectively. CONCLUSIONS: We report a highly reproducible, quantitative tool to objectively analyze vaginal flora that uses cutoff values for the concentrations of A. vaginae and G. vaginalis to establish the molecular diagnosis of BV.
Assuntos
Actinobacteria/isolamento & purificação , Contagem de Colônia Microbiana/métodos , Gardnerella vaginalis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Vaginose Bacteriana/diagnóstico , Actinobacteria/genética , Candida albicans/genética , Candida albicans/isolamento & purificação , Primers do DNA/genética , Feminino , Gardnerella vaginalis/genética , Humanos , Lactobacillus/genética , Lactobacillus/isolamento & purificação , Mobiluncus/genética , Mobiluncus/isolamento & purificação , Mycoplasma hominis/genética , Mycoplasma hominis/isolamento & purificação , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Ureaplasma urealyticum/genética , Ureaplasma urealyticum/isolamento & purificaçãoRESUMO
We report the first case of mother-to-child transmission of human immunodeficiency virus type 1 strains harboring multiple mutations including the Q151M multinucleoside reverse transcriptase inhibitor resistance mutation. Sustained virologic success was obtained in the infant with postnatal antiretroviral therapy optimized according to genotypic drug resistance testing and therapeutic drug monitoring.
Assuntos
Farmacorresistência Viral/genética , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/genética , Transmissão Vertical de Doenças Infecciosas , Adulto , Substituição de Aminoácidos/genética , Antirretrovirais/uso terapêutico , Feminino , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/isolamento & purificação , Humanos , Recém-Nascido , GravidezRESUMO
Development of hepatitis B virus (HBV)-resistant strains following nucleos(t)ide analogue treatment is a major concern. The A181V mutation within the reverse transcriptase (RT) of HBV has been shown to be associated with HBV resistance to adefovir dipivoxil (ADV), and its level of sensitivity to other nucleos(t)ide analogues is an important issue. This article reports two cases of chronically HBV infected patients who developed rtA181V HBV mutants following lamivudine (LAM) monotherapy. This was subsequently associated with virological breakthrough under LAM monotherapy or LAM/ADV bi-therapy, which were rescued by tenofovir disoproxil fumarate treatment. These observations suggest that rtA181V mutation may unusually emerge under LAM monotherapy, and may be associated with cross resistance to LAM and ADV, but remains sensitive to tenofovir disoproxil fumarate. Moreover, they highlight that HBV sequence analysis is an essential tool to optimize therapeutic management of HBV chronic infection in clinical practice in order to choose the appropriate nucleos(t)ide analogues.
Assuntos
Adenina/análogos & derivados , Farmacorresistência Viral Múltipla , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Lamivudina/uso terapêutico , Organofosfonatos/farmacologia , Adenina/farmacologia , Adulto , Antivirais/uso terapêutico , DNA Viral/sangue , Esquema de Medicação , Humanos , Masculino , Mutação , Seleção GenéticaRESUMO
OBJECTIVE: To determine to which extent the mutations K65R, L74V/I and T215Y/F are linked to the same HIV-1 genome. METHODS: Retrospective analysis of the Marseille database (8866 sequences from 3720 patients). The HIV-1 pol gene from four patients' viruses harbouring the double mutant (pure species or genotypic mixtures) was amplified, cloned and sequenced. RESULTS: The four patients had a mean viral load of 5.6 log(10)copies/ml. Analysis of 73 clones (patient 1: 12 clones; patient 2: 27 clones; patient 3: 13 clones; patient 4: 21 clones) showed that 29 clones harboured K65R and L74V/I mutations. Twenty-three per cent of clones from the two bulk sequences harbouring K65K/R and T215T/Y genotypic mixtures contained K65R and T215Y on the same viral genome. CONCLUSIONS: The co-linearity of 65R and 74V or 65R and 215Y amino-acids on the same genome is rare. A high viral load (6.19 log(10)) associated with the coexistence of 65R and 74V on the same HIV-1 genome suggests possible compensatory mechanisms.
Assuntos
Bases de Dados Genéticas , Genoma Viral , Transcriptase Reversa do HIV/genética , HIV-1/genética , HIV-1/enzimologia , Humanos , Mutação , Estudos Retrospectivos , Carga ViralRESUMO
We report a case of hepatitis B virus reactivation that occurred after immunosuppressive treatment in a hepatitis B surface antigen-negative patient with detectable antibodies to hepatitis B core antigen and hepatitis B surface antigen. Sequence analysis revealed an 11-bp insertion in the core promoter region of hepatitis virus DNA, creating a novel hepatocyte nuclear factor-1 binding site. This led to enhanced viral replication and fulminant hepatitis.
Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B/tratamento farmacológico , Falência Hepática Aguda/tratamento farmacológico , Regiões Promotoras Genéticas/genética , Feminino , Hepatite B/virologia , Anticorpos Anti-Hepatite B/sangue , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/fisiologia , Humanos , Imunossupressores/uso terapêutico , Falência Hepática Aguda/virologia , Dados de Sequência Molecular , Mutação , Ativação ViralRESUMO
BACKGROUND: In Africa, relapsing fever borreliae are neglected arthropod-borne pathogens causing mild to deadly septicemia and miscarriage. The closely related Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis and Borrelia hispanica are rarely diagnosed at the species level, hampering refined epidemiological and clinical knowledge of the relapsing fevers. It would be hugely beneficial to have simultaneous detection and identification of Borrelia to species level directly from clinical samples. METHODOLOGY/PRINCIPAL FINDINGS: We designed a multiplex real-time PCR protocol targeting the 16S rRNA gene detecting all four Borrelia, the glpQ gene specifically detecting B. crocidurae, the recN gene specifically detecting B. duttonii/B. recurrentis and the recC gene specifically detecting B. hispanica. Compared to combined 16S rRNA gene and flaB gene sequencing as the gold standard, multiplex real-time PCR analyses of 171 Borrelia-positive and 101 Borrelia-negative control blood specimens yielded 100% sensitivity and specificity for B. duttonii/B. recurrentis and B. hispanica and 99% sensitivity and specificity for B. crocidurae. CONCLUSIONS/SIGNIFICANCE: The multiplex real-time PCR developed in this study is a rapid technique for both molecular detection and speciation of relapsing fever borreliae from blood in Africa. It could be incorporated in point-of-care laboratory to confirm diagnosis and provide evidence of the burden of infection attributed to different species of known or potentially novel relapsing fever borreliae.
Assuntos
Borrelia/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Febre Recorrente/diagnóstico , África , Técnicas Bacteriológicas/métodos , Sangue/microbiologia , Borrelia/classificação , Borrelia/genética , Humanos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
A 16S rDNA sequence-based investigation of methanogenic Archaea in the human stools found Methanobrevibacter smithii in 99.2% and Methanosphaera stadtmanae in 32.6%. The recently described Methanomassiliicoccus luminyensis found by others to be representative of a new order of methanogenic Archaea was found in 4% of stool specimens. The prevalence of M. luminyensis significantly increased with age, contrary to M. smithii and M. stadtmanae.
Assuntos
Trato Gastrointestinal/microbiologia , Metagenoma , Methanobacteriaceae/genética , Methanobrevibacter/genética , RNA Ribossômico 16S/genética , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Primers do DNA , DNA Arqueal/análise , Fezes/microbiologia , Humanos , Lactente , Recém-Nascido , Methanobacteriaceae/classificação , Methanobacteriaceae/isolamento & purificação , Methanobrevibacter/classificação , Methanobrevibacter/isolamento & purificação , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase em Tempo RealAssuntos
Anticorpos Antivirais/imunologia , Infecção Hospitalar/imunologia , Antígenos da Hepatite B/imunologia , Hepatite B/imunologia , Falência Renal Crônica/imunologia , Infecção Hospitalar/epidemiologia , França/epidemiologia , Vacinas contra Hepatite B , Humanos , Incidência , Diálise Renal/efeitos adversos , Fatores de RiscoRESUMO
BACKGROUND: Identification of the Mycobacterium tuberculosis complex organisms to the species level is important for diagnostic, therapeutic and epidemiologic perspectives. Indeed, isolates are routinely identified as belonging to the M. tuberculosis complex without further discrimination in agreement with the high genomic similarity of the M. tuberculosis complex members and the resulting complex available identification tools. FINDINGS: We herein develop a pyrosequencing assay analyzing polymorphisms within glpK, pykA and gyrB genes to identify members of the M. tuberculosis complex at the species level. The assay was evaluated with 22 M. tuberculosis, 21 M. bovis, 3 M. caprae, 3 M. microti, 2 M. bovis BCG, 2 M. pinnipedii, 1 M. canettii and 1 M. africanum type I isolates. The resulted pyrograms were consistent with conventional DNA sequencing data and successfully identified all isolates. Additionally, 127 clinical M. tuberculosis complex isolates were analyzed and were unambiguously identified as M. tuberculosis. CONCLUSION: We proposed a pyrosequencing-based scheme for the rapid identification of M. tuberculosis complex isolates at the species level. The assay is robust, specific, rapid and can be easily introduced in the routine activity.
RESUMO
Probiotic food is manufactured by adding probiotic strains simultaneously with starter cultures in fermentation tanks. Here, we investigate the accuracy and feasibility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for bacterial identification at the species level in probiotic food and yoghurts. Probiotic food and yoghurts were cultured in Columbia and Lactobacillus specific agar and tested by quantitative real-time PCR (qPCR) for the detection and quantification of Lactobacillus sp. Bacterial identification was performed by MALDI-TOF analysis and by amplification and sequencing of tuf and 16S rDNA genes. We tested 13 probiotic food and yoghurts and we identified by qPCR that they presented 10(6) to 10(7) copies of Lactobacillus spp. DNA/g. All products contained very large numbers of living bacteria varying from 10(6) to 10(9) colony forming units/g. These bacteria were identified as Lactobacillus casei, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus delbrueckii, and Streptococcus thermophilus. MALDI-TOF MS presented 92% specificity compared to the molecular assays. In one product we found L. lactis, instead of Bifidus spp. which was mentioned on the label and for another L. delbrueckii and S. thermophilus instead of Bifidus spp. MALDI-TOF MS allows a rapid and accurate bacterial identification at the species level in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified. Practical Application: MALDI-TOF MS is rapid and specific for the identification of bacteria in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified.
Assuntos
Microbiologia de Alimentos , Probióticos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Iogurte/microbiologia , Bifidobacterium/classificação , Bifidobacterium/isolamento & purificação , Contagem de Colônia Microbiana , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Fermentação , Alimento Funcional/microbiologia , Lacticaseibacillus casei/classificação , Lacticaseibacillus casei/isolamento & purificação , Lactobacillus delbrueckii/classificação , Lactobacillus delbrueckii/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Streptococcus thermophilus/classificação , Streptococcus thermophilus/isolamento & purificaçãoRESUMO
BACKGROUND: The worldwide expanding Mycobacterium tuberculosis W-Beijing family is associated with treatment failure and relapse. Its identification currently relies on spoligotyping and conventional sequencing. We developed pyrosequencing as an alternative method for its identification. FINDINGS: Pyrosequencing found a G/A substitution in the Rv0927c-pstS3 intergenic spacer and a RD105 deletion, identifying 8/104 M. tuberculosis isolates as W-Beijing isolates. In addition, pyrosequencing found a previously unreported TGC deletion in the Rv0927c gene of W-Beijing isolates. Total concordance was found between the pyrosequencing data and conventional sequencing, as well as reference molecular identification. Multispacer Sequence Typing assigned the W-Beijing isolates to the Asian lineage and the 96 non-W-Beijing isolates to the Euro-American lineage (P < 10-5). The W-Beijing isolates were all susceptible to streptomycin, rifampin, isoniazid, ethambutol, and pyrazinamide; no resistance-associated mutations were detected in these eight W-Beijing isolates. There were no statistically significant differences in the antibiotic susceptibility of W-Beijing and non-W-Beijing isolates (p = 0.2, X2 test). Pyrosequencing correctly identified M. tuberculosis organisms in 26/26 sputum specimens exhibiting acid-fast bacilli. Pyrosequencing results were obtained within four hours, incurring an estimated cost of 1.86 euro/test. CONCLUSION: Pyrosequencing of the Rv0927c gene and adjacent intergenic spacer is an efficient, low-cost technique for the rapid identification of W-Beijing isolates.