Changes of gut microflora in newly diagnosed IgA nephropathy patients and its correlation with clinical risk factors / 北京大学学报(医学版)

OBJECTIVE@#To investigate the gut microbiota in newly diagnosed IgA nephropathy patients with chronic kidney disease (CKD) stages 1-2 and the association between the gut microbiota and the clinical risk factors of IgA nephropathy.@*METHODS@#Fresh fecal samples were collected from nineteen newly diagnosed IgA nephropathy patients with CKD stages 1-2 and fifteen age- and sex-matched healthy controls. Fecal bacterial DNA was extracted and microbiota composition were characterized using 16S ribosomal RNA (16S rRNA) high-throughput sequencing for the V3-V4 region. The Illumina Miseq platform was used to analyze the results of 16S rRNA high-throughput sequencing of fecal flora. At the same time, the clinical risk factors of IgA nephropathy patients were collected to investigate the association between the gut microbiota and the clinical risk factors.@*RESULTS@#(1) At the phylum level, the abundance of Bacteroidetes was significantly reduced (P=0.046), and the abundance of Actinobacteria was significantly increased (P=0.001). At the genus level, the abundance of Escherichia-Shigella, Bifidobacte-rium, Dorea and others were significantly increased (P < 0.05). The abundance of Lachnospira, Coprococcus_2 and Sutterella was significantly reduced (P < 0.05). (2) There was no significant difference in the abundance of gut microbiota between the newly diagnosed IgA nephropathy patients and the healthy control group (P>0.05), but there were differences in the structure of the gut microbiota between the two groups. The results of LEfSe analysis showed that there were 16 differential bacteria in the newly diagnosed IgA nephropathy patients and healthy controls. Among them, the abundance of the newly diagnosed IgA nephropathy patients was increased in Enterobacteriales, Actinobacteria, Escherichia-Shigella, etc. The healthy control group was increased in Bacteroidetes and Lachnospira. (3) The result of redundancy analysis (RDA) showed that Bifidobacterium was positively correlated with serum IgA levels, 24-hour urinary protein levels and the presence of hypertension. Lachnoclostridium was positively correlated with the presence of hypertension. Escherichia-Shigella was positively correlated with urine red blood cells account. Bifidobacterium was positively correlated with the proliferation of capillaries. Faecalibacterium was positively correlated with cell/fibrocytic crescents. Ruminococcus_2 was positively correlated with mesangial cell proliferation, glomerular segmental sclerosis and renal tubular atrophy/interstitial fibrosis.@*CONCLUSION@#The gut microbiota in the newly diagnosed IgA nephropathy patients with CKD stages 1-2 is different from that of the healthy controls. Most importantly, some gut bacteria are related to the clinical risk factors of IgA nephropathy. Further research is needed to understand the potential role of these bacteria in IgA nephropathy.

Analysis of the nail microbiome in patients with onychomycosis by high-throughput sequencing of 16S rDNA and internal transcribed spacer regions / 中华皮肤科杂志

Objective:To investigate differences in bacterial and fungal microbiome between infected nails and healthy nails among patients with onychomycosis.Methods:Nail scraping samples were collected from infected nails and healthy nails of 31 patients with onychomycosis, who visited Dalian Dermatosis Hospital from August 2020 to July 2021. The total DNA of nail microbiota was extracted, and the V3-V4 regions of the bacterial 16S rDNA gene and the fungal internal transcribed spacer (ITS) region were amplified and sequenced using Illumina technology. The USEARCH and mothur softwares were used for data cluster analysis to obtain the operational taxonomic units (OTUs) , Wilcoxon rank sum test was used to analyze α diversity, analysis of similarities (ANOSIM) was performed to analyze β diversity, linear discriminant analysis of effect size (LEfSe) was performed to evaluate the species difference.Results:Among the 31 patients with onychomycosis, 16 were males and 15 were females. According to the age, they were divided into young group (18 - 35 years old, 10 cases) , middle-aged group (36 - 60 years old, 11 cases) , and elderly group (over 60 years old, 10 cases) . As the α-diversity analysis revealed, the infected nail group showed significantly decreased Shannon index ( W = 290, P = 0.007) , but significantly increased Simpson index ( W = 663, P = 0.010) compared with the healthy nail group, suggesting that the diversity and evenness of bacterial communities were lower in the infected nail group than in the healthy nail group; however, there was no significant difference in the diversity of fungal communities between the infected nail group and healthy nail group. The β-diversity analysis based on the unweighted-UniFrac distance matrix showed no significant difference in the fungal or bacterial community composition between the infected nail group and healthy nail group (bacterial communities: R = 0.0052, P = 0.331; fungal communities: R = 0.0036, P = 0.337) ; the β-diversity analysis based on the weighted-UniFrac distance matrix showed significant differences in the abundance of bacterial and fungal communities between the two groups (both P = 0.001) . In terms of the species composition, the bacterial flora with significantly decreased abundance in the infected nail group compared with the healthy nail group included Bacteroidetes, Proteobacteria, Betaproteobacteria, Burkholderiales, Ralstonia, Sphingomonas and Streptococcus, while the abundance of Bacilli, Bacillales and Staphylococcus was significantly higher in the infected nail group than in the healthy nail group. Compared with the healthy nail group, the fungal flora with significantly increased abundance in the infected nail group included Eurotiomycetes, Onygenales, Leotiomycetes-ord-incertae-sedis, Arthrodermataceae, Periconia, Erysiphe, Tilletiopsis, Trichophyton, Erysiphe cruciferarum, Trichophyton rubrum, Malassezia sympodialis, while the abundance of Saccharomycetes, Saccharomycetales, Saccharomycetaceae, Dothioraceae, Candida and Alternaria was significantly lower in the infected nail group than in the healthy nail group. Conclusion:The diversity and abundance of bacterial communities significantly differed between infected nails and healthy nails among patients with onychomycosis, while only the abundance of fungal communities differed between the two groups, and perhaps there was correlations between some bacterial and fungal communities.

Polymerase chain reaction targeting 16S ribosomal RNA for the diagnosis of bacterial meningitis after neurosurgery

OBJECTIVES: Bacterial and aseptic meningitis after neurosurgery can present similar clinical signs and symptoms. The aims of this study were to develop and test a molecular method to diagnose bacterial meningitis (BM) after neurosurgery. METHODS: A 16S ribosomal RNA gene PCR-based strategy was developed using artificially inoculated cerebrospinal fluid (CSF) followed by sequencing. The method was tested using CSF samples from 43 patients who had undergone neurosurgery and were suspected to suffer from meningitis, and from 8 patients without neurosurgery or meningitis. Patients were classified into five groups, confirmed BM, probable BM, possible BM, unlikely BM, and no meningitis. RESULTS: Among the samples from the 51 patients, 21 samples (41%) were culture-negative and PCR-positive. Of these, 3 (14%) were probable BM, 4 (19%) were possible BM, 13 (62%) were unlikely BM, and 1 (5%) was meningitis negative. Enterobacterales, non-fermenters (Pseudomonas aeruginosa and Acinetobacter baumannii), Staphylococcus haemolyticus, Granulicatella, Variovorax, and Enterococcus cecorum could be identified. In the group of patients with meningitis, a good agreement (3 of 4) was observed with the results of cultures, including the identification of species. CONCLUSION: Molecular methods may complement the diagnosis, guide treatment, and identify non-cultivable microorganisms. We suggest the association of methods for suspected cases of BM after neurosurgery, especially for instances in which the culture is negative.

Analysis of differences in gut microbiota in patients with chronic spontaneous urticaria by 16S rRNA high-throughput sequencing / 中华皮肤科杂志

Objective:To investigate differences in gut microbiota between patients with chronic spontaneous urticaria (CSU) and healthy controls.Methods:A total of 18 patients with CSU (CSU group) and 18 age- and gender-matched healthy controls (HC group) were enrolled from Department of Dermatology, Tianjin First Central Hospital between January 2019 and December 2019. Fecal samples were collected from these subjects, and total DNA was extracted. The 16S rRNA sequencing technology was used to identify microbial species in gut microbiota, and bioinformatics methods were applied to analyze differences in gut microbiota composition between the 2 groups. The SPSS 23.0 software was used for statistical analysis of the experimental data.Results:In terms of α diversity, there was no significant difference in the Observed OTU index, Chao1 index, Shannon index or Simpson index between the CSU group (161.28 ± 35.47, 161.31 ± 35.51, 5.15 ± 0.47, 0.94 ± 0.03, respectively) and HC group (154.89 ± 54.46, 154.92 ± 54.43, 4.92 ± 0.88, 0.91 ± 0.08, respectively; t = 0.417, 0.417, 0.952, 1.116, respectively, all P > 0.05) . In terms of β diversity, principal component analysis showed that the first and second principal components explained 6.66% and 4.93% respectively, and there was no significant difference in the microbiota structure between the 2 groups ( P = 0.672) . The relative abundance of the genus Holdemania in the gut microbiota significantly differed between the CSU group and HC group (0.04% vs. 0.01%, P = 0.025) . Conclusion:The gut microbiota differs between the patients with CSU and healthy controls.

Analysis of gut microbiota change in colorectal adenomatous polyps patients by 16S rRNA sequencing / 中华检验医学杂志

Objective@#To explore the characteristics of gut microbiota change in colorectal adenomatous polyps (CAP), which has been considered as precancerous lesion for colorectal cancer.@*Methods@#Thirty patients with colon adenomatous polyps (CAP group) and thirty healthy individuals without adenomatous polyps (HC group) who underwent colonoscopy at the First Affiliated Hospital of Kunming Medical University from November 2017 to April 2018 were randomly collected. The biopsy mucosae were collected by endoscopic electrocoagulation, and DNA was extracted to amplify 16S rRNA V3-V4 region, followed high-throughput sequencing with Illumina MiSeq platform. The experimental results were analyzed using Wilcoxon test.@*Results@#The alpha diversity of CAP patients was higher than that of healthy controls (Chao & Ace P<0.01). A decreased abundance of Bacteroidetes (FC=0.38) was observed at phylum level(P<0.05). At genus level, the abundances of Bacteroides (FC=0.32) , Escherichia (FC=0.57) , Ruminococcus (FC=0.42) , Blautia (FC=0.27) , and Dorea (FC=0.57) were decreased (P<0.05), but those of Pseudomonas(FC=2.43), Lactococcus(FC=2.84), Geobacillus(FC=2.07), and Acinetibacter(FC=2.36) were increased in CAP patients (P<0.05).@*Conclusions@#Compared with healthy volunteers, there are significant differences in the abundance and diversity of the adenoma tissue in CAP patients, indicating that there is an imbalance of gut microbiota in the adenomatous polyps. The imbalance of intestinal microenvironment may contribute to the occurrence and development of CAP.

Difference Analysis Based on 16S rRNA Sequencing of Different Soil Bacterial Communities / 法医学杂志

Objective To study the structure and differences of bacterial communities in different soils, and to explore the effectiveness of 16S rRNA sequencing in identification of different soil. Methods Soil samples from 7 places in Shanghai were collected, then bacterial genomic DNA were extracted from them. The fragments of hypervariable region from 16S rRNA sequences were sequenced with high-throughput sequencing techniques. The results were quantified or visualized with bioinformatics software. The differences in diversity and abundance among the three kinds of bacterial communities in soil samples from grassland, forests and beaches were compared and analyzed. Results The statistical differences that existed among the alpha diversity indexes of bacterial communities in soil samples of grassland, forests and beaches had statistical significance. The relative abundance and diversity of bacterial communities in these three kinds of soil were significantly different. Grassland soil had higher Acidobacteria abundance, forest soil had higher Proteobacteria abundance, beach soil had higher Actinobacteria abundance. However, the differences in soil bacterial communities in artificial grasslands, natural grasslands and industrial district grasslands did not have statistical significance. Conclusion 16S rRNA sequencing can effectively distinguish different soils. This method may be able to provide clues for first crime scene inference in criminal cases.

Progress in the 16S rRNA Gene Sequencing in Forensic Science / 法医学杂志

Forensic microorganism is one of the hotspots of forensic science research. Due to its conservatism and specificity, the 16S rRNA gene is found to be an ideal marker for forensic identification. With the rapid development of high throughput sequencing technology, the research on microorganisms has been gradually applied to many fields such as environment and health care. In the field of forensic science, the results of forensic microbiology research, represented by 16S rRNA gene sequencing, are also gradually applied to forensic practice, such as biological samples identification, individual identification, postmortem interval estimation, and regional inference, which not only provide clues for the investigation of cases but also complement and assist traditional methods. This paper describes the research methods and related sequencing technologies of 16S rRNA gene sequencing, summarizes its research progress, and discusses the application value and potential of 16S rRNA in forensic science.

Composition of gut microbiota varies in very low birth weight infants within six months after birth / 中华围产医学杂志

Objective To investigate the diversity of gut microbiota and its dynamic changes in very low birth weight infants (VLBWIs) during the first six months after birth.Methods From January to December in 2015,53 VLBWIs admitted to the Neonatal Intensive Care Unit (NICU) were recruited.Stool samples were collected from each subject on day 1,7,14 and 28 after birth as well as on day 180 during a follow-up visit.High-throughput 16S rRNA gene sequencing and bioinformatics analysis of bacterial DNA extracted from stool samples were performed using Illumina MiSeq platform double-end sequencing.Results (1) Phyla level:At all five time points,the dominant phyla were all Proteobacteria,Firmicutes,Bacteroidetes and Actinobacteria.The median relative abundance of Proteobacteria was 0.598 5 (0.122 3～0.942 6) on day 1,then rose to 0.893 2 (0.478 1～0.987 0) after one week,maintained at 0.943 2～0.966 0 within 28 days,and later dropped back to the same level as day 1 on day 180 (all P＜0.05).In contrast,the median relative abundance of Actinobacteria on day 180 was significantly higher than that on days 1,7,14 and 28 (all P＜0.05).(2) Genus level:The relative abundance of Klebsiella increased significantly between days 7 and 28 as compared with the lower level on day 1 [0.326 5～0.368 2 vs 0.003 1(0.000 8～0.026 0),all P＜0.05],but significantly decreased to a lower level on day 180 [0.008 1 (0.000 5～0.067 1)].There was no significant difference in the relative abundance of Escherichia within 14 days after birth.However,it significantly increased since day 28 (P＜0.05) and reached a peak on day 180 (P＜0.05).Infants were born with a certain abundance of Bifidobacterium [0.000 5 (0.000 1～0.004 2)],which remained at a very low level for 28 days before reaching to a higher level of 0.045 1 (0.010 2～0.124 8) on day 180 (P＜0.05).(3)The Shannon index on day 14 and 28 were lower than that of day 1 and day 180 (1.81±0.71,1.89±1.270.56 vs 2.33±1.29,2.2±0.5,all P＜0.05),respectively.Conclusions The diversity of gut microbiota in VLBWIs decreases during NICU hospitalization as compared with that at birth when Proteobacteria and Klebsiella becoming the dominant bacteria.However,the diversity was regained after discharge with the increase of Bifidobacterium,Escherichia and other residential bacteria,which indicates that NICU hospitalization is a risk factor for dysbiosis in VLBWIs.

The value of new 16S rRNA gene chip in pathogen detection of neonatal sepsis / 中国新生儿科杂志

Objective To study the value of new 16S rRNA gene chip in pathogen detection of neonatal sepsis.Method Newborns with suspected sepsis hospitalized in Peking University Shenzhen Hospital from January 2015 to December 2017 were chosen as the subjects.Blood culture and gene chip were both used to detect the pathogens of these infants.The positive rate,the detection time,and the blood volume needed for detection were compared between the two methods.Result A total of 306 cases of suspected neonatal sepsis were included in the study.Among them,34 (11.1％) were positive for blood culture and 54 (17.6％) were positive for gene chip.98 cases were diagnosed as neonatal sepsis,34 (34.7％) were positive for blood culture,and 52 (53.1％) were positive for gene chip.The positive rate of gene chip was higher than that of blood culture (P ＜ 0.05).For the 5 common pathogens of neonatal sepsis,the positive rate of gene chip was higher than that of blood culture.Time to positivity (TTP) and pathogen identification time of blood culture were (14.6 ± 5.5) h and (72.9 ± 19.0) h,respectively.TTP and pathogen identification time of gene chip were both 3 h.The detection time of gene chip was significantly less than that of blood culture (P ＜ 0.001).The blood volume needed for detection of blood culture and gene chip was 1 ～ 2 ml and 0.5 ml.Gene chip needs less blood volume than blood culture.Conclusion Compared with the traditional blood culture,gene chip can quickly detect the pathogens in the blood with higher positive rate and less blood volume.Gene chip is of great value in the diagnosis of neonatal sepsis.

Detection of 16S rRNA gene and biological characteristics of Mycobacterium abscessus / 中华皮肤科杂志

Objective To identify a clinical isolate of Mycobacterium abscessus.Methods A pus sample was collected from a patient with suspected nontuberculous mycobacterial infection who visited the Affiliated Hospital of Weifang Medical University on December 18,2017,and was subjected to bacterial culture,Gram staining and acid-fast staining.Drug sensitivity test was conducted by the proportion method.The genome DNA of the strain was extracted and amplified by PCR with the universal primer of 16S rRNA.The PCR products were sequenced after collection and purification,and were compared with the known sequence of Mycobacterium abscessus in GenBank database.The isolate was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).Results The clinical isolate was identified as Mycobacterium abscessus both by MALDI-TOF MS and 16S rRNA gene sequencing.The drug sensitivity test showed that the strain was sensitive to amikacin,moxifloxacin,levofloxacin,but was resistant to streptomycin,isoniazid,rifampicin,ethambutol,ofloxacin,kanamycin,capreomycin,aminosalicylic acid,protionamide and rifabutin.The patient was diagnosed with subcutaneous soft tissue infection in the left knee joint.According to the results of drug sensitivity test,the patient was treated with amikacin and levofloxacin,and her condition was improved after treatment.Conclusion The 16S rRNA gene detection and MALDI-TOF MS both can be applied in the identification of Mycobacterium abscessus.

Characterization of the fecal microbiota differs between age groups in Koreans

BACKGROUND/AIMS: Tens of trillions of microorganisms constitute the gut microbiota of the human body. The microbiota plays a critical role in maintaining host immunity and metabolism. Analyses of the gut microbial composition in Korea are limited to a few studies consisting of small sample sizes. To investigate the gut microbial community in a large sample of healthy Koreans, we analyzed the 16S ribosomal RNA of 4 representative bacterial genera Lactobacillus, Bifidobacterium, Bacteroides, and Clostridium. METHODS: A total of 378 DNA samples extracted from 164 infants and 214 adults were analyzed using quantitative real-time polymerase chain reaction. RESULTS: Analysis of 16S ribosomal RNA of 4 representative bacterial genera Lactobacillus, Bifidobacterium, Bacteroides, and Clostridium showed that the gut microbiota in infants had higher relative abundances of Bifidobacterium and Lactobacillus than that in adults, which was dominated by Bacteroides and Clostridium. CONCLUSIONS: To the best of our knowledge, this was the first study evaluating the distinct characteristics of the microbial community of Korean infants and adults. The differences between the 2 populations suggest that external factors such as age, diet, and the environment are important contributing factors to the change in gut microbial composition during development.

Native Valve Endocarditis due to Corynebacterium striatum confirmed by 16S Ribosomal RNA Sequencing: A Case Report and Literature Review

Corynebacterium species are non-fermentous Gram-positive bacilli that are normal flora of human skin and mucous membranes and are commonly isolated in clinical specimens. Non-diphtheriae Corynebacterium are regarded as contaminants when found in blood culture. Currently, Corynebacterium striatum is considered one of the emerging nosocomial agents implicated in endocarditis and serious infections. We report a case of native-valve infective endocarditis caused by C. striatum, which was misidentified by automated identification system but identified accurately by 16S ribosomal RNA sequencing, in a 55-year-old male patient. The patient had two mobile vegetations on his mitral valve, both of which had high embolic risk. Through surgical valve replacement and an antibiotic regimen, the patient recovered completely. In unusual clinical scenarios, C. striatum should not be simply dismissed as a contaminant when isolated from clinical specimens. The possibility of C. striatum infection should be considered even in an immunocompetent patient, and we suggest a genotypic assay, such as 16S rRNA sequencing, to confirm species identity.

Lung Microbiome Analysis in Steroid-Naїve Asthma Patients by Using Whole Sputum / 결핵및호흡기질환

BACKGROUND: Although recent metagenomic approaches have characterized the distinguished microbial compositions in airways of asthmatics, these results did not reach a consensus due to the small sample size, non-standardization of specimens and medication status. We conducted a metagenomics approach by using terminal restriction fragment length polymorphism (T-RFLP) analysis of the induced whole sputum representing both the cellular and fluid phases in a relative large number of steroid naïve asthmatics. METHODS: Induced whole sputum samples obtained from 36 healthy subjects and 89 steroid-naїve asthma patients were analyzed through T-RFLP analysis. RESULTS: In contrast to previous reports about microbiota in the asthmatic airways, the diversity of microbial composition was not significantly different between the controls and asthma patients (p=0.937). In an analysis of similarities, the global R-value showed a statistically significant difference but a very low separation (0.148, p=0.002). The dissimilarity in the bacterial communities between groups was 28.74%, and operational taxonomic units (OTUs) contributing to this difference were as follows: OTU 789 (Lachnospiraceae), 517 (Comamonadaceae, Acetobacteraceae , and Chloroplast), 633 (Prevotella), 645 (Actinobacteria and Propionibacterium acnes), 607 (Lactobacillus buchneri, Lactobacillus otakiensis, Lactobacillus sunkii, and Rhodobacteraceae), and 661 (Acinetobacter, Pseudomonas, and Leptotrichiaceae), and they were significantly more prevalent in the sputum of asthma patients than in the sputum of the controls. CONCLUSION: Before starting anti-asthmatic treatment, the microbiota in the whole sputum of patients with asthma showed a marginal difference from the microbiota in the whole sputum of the controls.

Evaluation of MALDI Biotyper and VITEK MS for identifying clinical isolated Streptococcus spp / 中华检验医学杂志

Objective Using 16S rRNA gene sequencing as the gold standard method,to compare the performance of two matrix-assisted laser desorption ionization time of flight mass spectrometry system (MALDI Biotyper and VITEK MS) for identifying clinical isolates of Streptococcus spp.Methods One hundred and sixty two clinical Streptococcus isolates were collected at the Second Affiliated Hospital of Zhejiang University,from April to June,2014,and confirmed by 16S rRNA gene sequencing analysis.MALDI Biotyper and VITEK MS mass spectrometry system were used for identification and further evaluated by performance respectively.Results Of all the isolates tested,155 (155/162,95.68％) Streptococcus isolates were accurately identified to species level by MALDI Biotyper.Besides,MALDI Biotyper identified three Streptococcus mitis group as S.pneumoniae and one S.parasanguinis as S.australis.Another three S.pneumonia isolates were not identified accurately (values ＜ 1.7).Although 156 (156/162,96.30％) isolates were accurately identified to species level (including subspecies) by VITEK MS system,two S.pneumoniae as S.mitis/S.oralis and one S.euinus as S.infantarius ssp.infantarius were misidentified.The two systems showed a 100％ (51/51) accuracy in identifying all S.pyogenes and S.agalactiae isolates,and an accuracy higher than 85％ for S.pneumoniae.Conclusions Both systems showed potent identification ability for Streptococcus spp.,VITEK MS system showed more clinical significance in accurately identifying some subspecies.Mass spectrometry system can be used as a rapid identification method for Streptococcs spp.in clinical practice.

Acute Cellulitis Caused by Neisseria skkuensis / 대한임상미생물학회지

A 73-year-old man visited our hospital because of pain with swelling and redness on the right foot dorsum. He was diagnosed with liver cirrhosis and nodular hepatic cellular carcinoma. Lower extremity CT scan and MRI showed abscess formation in the right foot dorsum. Gram-negative cocci were recovered from the culture of drained pus at the site and identified as Neisseria skkuensis by 16S rRNA gene sequencing. Here, we report the first case of cellulitis due to N. skkuensis and provide a literature review.

Desenvolvimento de um marcador molecular para o diagnóstico e monitoramento da sepse neonatal bacteriana / Development of a molecular marker for diagnosis and monitoring of neonatal bacterial sepsis

A sepse bacteriana constitui a causa mais frequente de óbitos neonatais, e seu diagnóstico é complexo devido à inexistência de um teste laboratorial definitivo. O presente estudo desenvolveu uma técnica de amplificação quantitativa (qPCR) do gene 16S rDNA de bactérias tanto para o diagnóstico de sepse neonatal, quanto para avaliar se a qPCR é capaz de monitorar o tratamento. Para ser recrutado o RN deveria apresentar ao menos dois sinais/sintomas sugestivos de sepse, e dois parâmetros laboratoriais alterados. Amostras de sangue foram colhidas no tempo zero (suspeita de sepse), 48 horas e sete dias após o início da antibioticoterapia. Foram analisados 73 RN (21 RNT e 52 RNPT) com suspeita de sepse neonatal. A hemocultura foi positiva em 32 RN (43,8% - sepse confirmada) e negativa em 41 (56,2% - sepse clínica), enquanto a qPCR foi positiva em 65 RN (89%) e negativa em oito casos (11%). Dentre os 32 RN com sepse confirmada (11 RNT e 21 RNPT), neutrofilia foi encontrada em 22 (68,75%), CRP elevada em 21 (65,62%), plaquetopenia em 15 (46,87%) e leucopenia em 14 (43,75%). Foram analisadas 200 amostras dos 73 casos suspeitos, considerando os três tempos de coleta, resultando em 36 hemoculturas positivas (18,0%) e 135 qPCR positivas (67,5%). Nas 36 hemoculturas positivas houve 38 isolamentos. Bactérias Gram-positivas foram encontradas em 32 amostras (84,21%) e Gram-negativas em seis (15,78%). Staphylococcus coagulase negativa predominou dentre as Gram-positivas (75,0%). No grupo de 32 RN com sepse confirmada a qPCR foi positiva em 30 (30/32 - 93,7%). Em 14 casos (47%) a qPCR antecipou o diagnóstico de sepse quando comparada à hemocultura e foi positiva no tempo zero em 22 casos (68,75%), enquanto a hemocultura foi positiva em 11. Dos 41 casos de sepse clínica, a qPCR foi positiva em 35 (85,4%); em 26 casos (74,3%) já no tempo zero. O teste de McNemar encontrou discordância entre os resultados das hemoculturas e qPCR (p<0,0001, IC de 95%), indicando...

Bacterial sepsis constitutes one of the most frequent causes of neonatal deaths and its diagnosis is difficult due to the lack of a definitive laboratorial approach. The present study developed a bacterial 16S rDNA-based quantitative real time polymerase chain reaction (qPCR) both to the diagnosis of neonatal sepsis and to evaluate if qPCR is capable of monitoring antimicrobial treatment. For enrollment, the newborn (NB) should present, at least, two signs/symptoms suggestive of sepsis, and two abnormal laboratory parameters. Blood samples were collected on day zero (suspected sepsis), 48 hours and 7 days after the initiation of antibiotic therapy. Seventy-three newborns with suspected sepsis were recruited (21 term NB and 52 preterm NB), blood culture was positive in 32 (43.8% - confirmed sepsis) and negative in 41 (56.2% - clinical sepsis), while qPCR was positive in 65 (89.0%) and negative in 8 cases (11.0%). Considering the group of 32 NB with confirmed sepsis (11 TNB and 21PTNB), qPCR was positive in 30 (30/32 - 93.7%). Neutrophilia was found in 22 NB (68.75%), elevated CRP in 21 (65.62%), thrombocytopenia in 15 (46.87%) and leukopenia in 14 (43.75%). Of the 73 cases, taking into account the three collected samples (day zero, 48h and 7 days), 200 samples were analyzed, with 36 positive blood culture (18.0%) and 135 positive qPCR (67.5%). Of the 36 positive blood cultures, there were 38 bacterial isolations. Gram-positive bacteria were found in 32 samples (84.21%) and Gram-negative in 6 (15.78%). Coagulase-negative Staphylococcus was predominant in the Grampositive group (75.0%). In 14 cases, qPCR anticipated the diagnosis when compared with blood culture, and was positive in 22 cases on day zero (68.75%), whereas blood culture was positive in 11. Among the 41 cases of clinical sepsis, qPCR was positive in 35 (85.4%); of these 26 (74.3%) on day zero. McNemar test found discordance between the results of blood cultures and qPCR (p < 0.0001, CI of 95%)...

Rapid identification of Nontuberculous Mycobacterium using matrix assisted laser desorption/ionization time of flight mass spectrometry / 中华检验医学杂志

Objective The purpose of this study was to compare and develop the method for identification of Non-tuberculous Mycobacteria (NTM) using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS),and evaluate the feasibility,accuracy and repeatability of MALDI-TOF MS to discriminate NTM.Methods Fifteen clinical strains were collected from January to March in 2012 and 68 environmental strains were retrospectively collected from 2011 to 2012.A protocol for sample pre-treatment and protein extraction was developed and utilized it to identify clinical and environmental isolates.The results from 16 s rRNA sequencing were served as control.Results Method A was more effective in protein extraction.Although all the three methods got the same species result,a total of 83 strains belonging to 10 distinct species grown in Middle brook 7H10 media were analyzed.All members of the NTM were identified accurately at the genus level and 80.7％ (67/83) of strains could be identified at the species level.Six strains were identified at the complex level.81.9％ (68/83) of NTM got high spectral scores.The identification of cultured colony could be completed in 1.5 hours.And it had good reproducibility.Conclusion MALDI-TOF MS can be used as a rapid and accurate method for identification of Mycobacteria in clinical microbiology laboratories,implying its good application prospects.

First case of Nocardia nova spinal abscess in an immunocompetent patient

Nocardia are a group of aerobic actinomycetes that are filamentous gram-positive, weakly acid-fast, and cause opportunistic infection in immunocompromised patients. Primary Nocardia infection mostly involves lung, skin and less commonly, the central nervous system (CNS). Among Nocardia CNS infections, spinal infection is extremely rare. We describe the first case of a spinal abscess caused by Nocardia nova in an immunocompetent patient who experienced a penetrating facial injury six months earlier. Nocardia species were isolated from intradural spinal abscesses and identified by 16S rRNA, hsp65 and secA1 sequence analyses. Surgical excision and treatment with amikacin, cefotaxime, and oral erythromycin was successful.

Rapid detection of clinical common bacteria using DNA microarray / 中华检验医学杂志

Objective To detect eight kinds of clinical common pathogenic bacteria by DNA microarray.Methods Eight kinds of common pathogenic bacteria,including Staphylococcus aureus,Pseudomonas aeruginosa,Klebsiella pneumoniae,Escherichia coli,Proteus mirabilis,Enterobacter aerogenes,Pseudomonas fluorescens,Shigella sonnei were collected.Universal primers were designed to amplify 16S rRNA gene fragment from the genomic DNA of the eight bacteria,and probes were designed in the highly variable regions.DNA microarray detection system was established and used for detection of colleted bacteria.A total of 50 samples were collected from the Zhongnan Hospital of Wuhan University,including 6 blood samples,32 sputum samples,9 feces samples and 3 bronchoscope lavage samples.DNA were extracted and detected by the established DNA microarray system.Results The desired fragments were well amplified by the self-designed universal primers.The selected probes had good detection results according to repeated detection.Of the 50 samples detected,pathgenic bacteria were accurately detected in 47 samples.Other three samples were not detected as those bacteria were not included in the chip.By optimizing the detection process,the results could be reported within 8 hours.Observation of probe signal attenuation indicated that even attenuated after 60 days,but the attenuation did not affect the results.Conclusion A microarray system was established for detection of clinical common bacteria accurately and quickly,which provided foundation for its clinical application.

Identification of the strain 1012 isolated from the sample of EQA of the Chinese National Center of Clinical Laboratory, and phylogenetic analysis of Actinomyces turicensisand related bacteria in the family Actinomycetaceae / 中华检验医学杂志

ObjectivesTo identified the strain 1012 from the National Center of Clinical Laboratory of China for microbe inter-laboratory quality assessment in 2010, and study the taxonomic status of strain 1012 and related species in the genus Actinomyces. Methods The bacterial traditional morphological characteristics, commercial API systems, and 16S rRNA gene sequence analysis were applied to identify the problematic culture of strain 1012. The phylogenetic tree based on the remote information of the prokaryotes systems was constructed to study the taxonomic status and evolutionary relationship of the genus Actinomyces and related species in the family Actinomycetaceae. Results Strain 1012 was determined as a kind of facuhative anaerobic,non-spot-forming,Gram-positive coryneformbacteria,which was identifiedto Actinomyces turicensis for the phenotypic biochemical characteristics of more than 60 items, The comparative study of 16S rRNA gene showed the strain 1012 with 99. 8％ similarities to Actinomyces turicensis, but only 90. 6％ to the type species of Actinomyces bovis in the genus Actinomyces. However, the comparative study of 16S rRNA gene showed the strain 1012 with only 90. 6％ homology to the type species of Actinomyces bovis in the genus Actinomyces. Further phylogenetic analysis showed that nine independent clusters were grouped in the family Actinomycetaceae, of which four clusters were separately represented the genera Varibaculum,Mobiluncus, Actinobaculum and Arcanobacterium, while other five clusters all were designated to the genus Actinomyces. The study showed strain 1012 was located in genus Ⅲ of Actinomyces, yet with a relatively long genetic distance to Actinomyces bovis. ConclusionThe genus Actinomyces may be reclassified as one genus Actinomyces sensu stricto and several new genera for the genotypic characteristics.