重症急性呼吸道感染患儿中人鼻病毒分型检测及流行病学分析

目的:调查重症急性呼吸道感染患儿中人鼻病毒(HRV)的感染情况,初步了解不同型别HRV流行病学和临床特点。方法:收集2008年5月至2010年3月北京儿童医院重症急性呼吸道感染住院患儿鼻咽抽吸物样本259份,采用巢式PCR法,先用鼻病毒5′UTR引物筛查样本中该病毒总的感染情况,再用针对VP4-VP2区域的引物对阳性样本进行分型检测;同时对阳性样本进行其他常见呼吸道病毒共感染检测与流行病学及临床特点分析。结果:259例样本中,5′UTR初筛HRV阳性89例(34.4%),与其他常见呼吸道病毒存在共感染54例(60.7%);可通过VP4-VP2分型测序确定39例HRV-A(48.1%)、4例HRV-B(4.9%)、38例HRV-C(46.9%);HRV-A感染率最高的季节分布是秋季;HRV-A和HRV-C型感染者在临床特点上无明显差别。结论:HRV是儿童重症急性呼吸道感染的重要病原之一,新发现的HRV-C基因型感染率与HRV-A相似,但HRV在儿童重症急性呼吸道感染中的病原学意义还须进一步确认。     

糖尿病酮症酸中毒并社区获得性肺炎临床分析

目的:探讨糖尿病酮症酸中毒合并社区获得性肺炎的临床特点、治疗方法,为临床预防和治疗提供方法。方法:对2013年1月~2014年11月入住我院呼吸科病房的12例糖尿病酮症酸中毒合并社区获得性肺炎患者的临床资料、治疗、转归进行回顾性分析。结果:糖尿病酮症酸中毒合并社区获得性肺炎的患者危险因素有:意识状态、肺部基础疾病、贫血、低蛋白血症、血糖水平及降糖药物使用情况、年龄。经充分补液、小剂量胰岛素消酮、控制血糖、抗感染、呼吸机辅助通气、纠正离子紊乱及加强对症支持治疗后患者均好转出院。结论:在糖尿病酮症酸中毒合并社区获得性肺炎患者的诊治过程中,控制血糖是治疗的基础,抗感染是治疗的关键,同时改善营养和其他器官的功能状态可明显提高治愈率和降低病死率。     

High Human Bocavirus Viral Load Is Associated with Disease Severity in Children under Five Years of Age

Human bocavirus (HBoV) is a parvovirus and detected worldwide in lower respiratory tract infections (LRTIs), but its pathogenic role in respiratory illness is still debatable due to high incidence of co-infection with other respiratory viruses. To determine the prevalence of HBoV infection in patients with LRTI in Shanghai and its correlation with disease severity, we performed a 3-year prospective study of HBoV in healthy controls, outpatients and inpatients under five years of age with X-ray diagnosed LRTIs. Nasopharyngeal aspirates were tested by PCR for common respiratory viruses and by real time PCR for HBoV subtypes 1–4. Nasopharyngeal swabs from healthy controls and serum samples and stools from inpatients were also tested for HBoV1-4 by real time PCR. Viral loads were determined by quantitative real time PCR in all HBoV positive samples. HBoV1 was detected in 7.0% of inpatients, with annual rates of 5.1%, 8.0% and 4.8% in 2010, 2011 and 2012, respectively. Respiratory syncytial virus (RSV) subtype A was the most frequent co-infection detected; HBoV1 and RSVA appeared to co-circulate with similar seasonal variations. High HBoV viral loads (>10⁶ copies/ml) were significantly more frequent in inpatients and outpatients than in healthy controls. There was a direct correlation of high viral load with increasing disease severity in patients co-infected with HBoV1 and at least one other respiratory virus. In summary, our data suggest that HBoV1 can cause LRTIs, but symptomatic HBoV infection is only observed in the context of high viral load.     

Molecular and Phenotypic Characterization of Staphylococcus epidermidis Isolates from Healthy Conjunctiva and a Comparative Analysis with Isolates from Ocular Infection

Staphylococcus epidermidis is a common commensal of healthy conjunctiva and it can cause endophthalmitis, however its presence in conjunctivitis, keratitis and blepharitis is unknown. Molecular genotyping of S. epidermidis from healthy conjunctiva could provide information about the origin of the strains that infect the eye. In this paper two collections of S. epidermidis were used: one from ocular infection (n = 62), and another from healthy conjunctiva (n = 45). All isolates were genotyped by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec), detection of the genes icaA, icaD, IS256 and polymorphism type of agr locus. The phenotypic data included biofilm production and antibiotic resistance. The results displayed 61 PFGE types from 107 isolates and they were highly discriminatory. MLST analysis generated a total of 25 STs, of which 11 STs were distributed among the ocular infection isolates and lineage ST2 was the most frequent (48.4%), while 14 STs were present in the healthy conjunctiva isolates and lineage ST5 was the most abundant (24.4%). By means of a principal coordinates analysis (PCoA) and a discriminant analysis (DA) it was found that ocular infection isolates had as discriminant markers agr III or agr II, SCCmec V or SCCmec I, mecA gene, resistance to tobramycin, positive biofilm, and IS256⁺. In contrast to the healthy conjunctiva isolates, the discriminating markers were agr I, and resistance to chloramphenicol, ciprofloxacin, gatifloxacin and oxacillin. The discriminant biomarkers of ocular infection were examined in healthy conjunctiva isolates, and it was found that 3 healthy conjunctiva isolates [two with ST2 and another with ST9] (3/45, 6.66%) had similar genotypic and phenotypic characteristics to ocular infection isolates, therefore a small population from healthy conjunctiva could cause an ocular infection. These data suggest that the healthy conjunctiva isolates do not, in almost all cases, infect the eye due to their large genotypic and phenotypic difference with the ocular infection isolates.     

北京地区人偏肺病毒膜表面糖蛋白G编码基因和特征的分析

为了解北京地区人偏肺病毒(hMPV)膜表面糖蛋白G编码基因的特征,提取2003年和2004年各6份hMPV阳性的临床呼吸道标本中的RNA,经用随机引物合成cDNA后,用特异性引物扩增G蛋白全基因,克隆至pBS-T载体中并进行测序。用生物软件与GenBank中hMPV的基因序列进行比较和种系进化分析。12株hMPV可以分为两个主要的进化簇1和2。每个进化簇还可以再分为不同的亚进化簇。3株(2003年)hMPV属于进化簇1;9株(3株2003年和6株2004年)hMPV属于进化簇2。这12株hMPV G蛋白基因的核苷酸长度在624~711nt,使用了两种不同的终止密码,编码的氨基酸长度为208~236 aa,蛋白质分子量为22.9~25.8kD。与进化簇2相比,进化簇1的3株hMPV都出现了3个核苷酸的缺失,但未改变读码框架。同一进化簇内的hMPV G蛋白基因核苷酸和氨基酸的同源性分别为95.7%~100%和91.8%~100%,不同进化簇间的核苷酸和氨基酸的同源性分别为56.3%~57.4%和34.3%~38.2%。疏水性分析表明这12株hMPV G蛋白是典型的Ⅱ型跨膜锚定蛋白,分别与两个进化簇代表株的疏水图一致。这些hMPV的G蛋白氨基酸的替换集中在胞外区。它们都含有高百分比的脯氨酸(P)、丝氨酸(S)和苏氨酸(T),含有相当数量的位于胞外区的O-连接糖基化位点。这些hMPV的G蛋白的N-连接糖基化位点数目和位置不尽相同。通过对G蛋白基因的分析显示,2003年和2004年北京地区流行的hMPV分属于两种不同的进化簇。基因分析显示hMPV的G蛋白是高糖基化的膜表面蛋白,具有与同属于副粘病毒科、肺病毒亚科的人呼吸道合胞病毒(hRSV)的G蛋白相似的基因特征,推测其功能和在感染免疫中的作用相当于hRSV的G蛋白,但有待进一步深入研究予以证实。     

Treatment of Severe Community-Acquired Pneumonia with Oral Amoxicillin in Under-Five Children in Developing Country: A Systematic Review

Objective

To assess the evidence regarding efficacy of oral amoxicillin compared to standard treatment for WHO-defined severe community acquired pneumonia in under-five children in developing country.

Design

Systematic review and meta-analysis of data from published Randomized trials (RCTs).

Data sources

MEDLINE (1970– July 2012) via PubMed, Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, Issue 7, July 2012), and EMBASE (1988– June 2012).

Methods

Eligible trials compared oral amoxicillin administered in ambulatory setting versus standard treatment for WHO-defined severe community acquired pneumonia in children under-five. Primary outcomes were proportion of children developing treatment failure at 48 hr, and day 6. GRADE criteria was used to rate the quality of evidence.

Results

Out of 281 full text articles assessed for eligibility, 5 trials including 12364 children were included in the meta-analysis. Oral amoxicillin administered either in hospital or community setting is effective in treatment of severe pneumonia and is not inferior to the standard treatment. None of the clinical predictors of treatment failure by 48 hr (very severe disease, fever and lower chest indrawing, and voluntary with-drawl and loss to follow up) was significant between the two groups. The clinical predictors of treatment failure that were significant by day 6 were very severe disease, inability to drink, change of antibiotic, and fever alone. The effect was almost consistent across the studies.

Conclusion

Though oral amoxicillin is effective in treatment of severe CAP in under-five children in developing country, the evidence generated is of low-quality. More trials with uniform comparators are needed in order to strengthen the evidence.     

Prevalence of Herpes and Respiratory Viruses in Induced Sputum among Hospitalized Children with Non Typical Bacterial Community-Acquired Pneumonia

Objective

Few comprehensive studies have searched for viruses in infants and young children with community-acquired pneumonia (CAP) in China. The aim of this study was to investigate the roles of human herpes viruses (HHVs) and other respiratory viruses in CAP not caused by typical bacterial infection and to determine their prevalence and clinical significance.

Methods

Induced sputum (IS) samples were collected from 354 hospitalised patients (infants, n = 205; children, n = 149) with respiratory illness (CAP or non-CAP) admitted to Wenling Hospital of China. We tested for HHVs and respiratory viruses using PCR-based assays. The epidemiological profiles were also analysed.

Results

High rate of virus detection (more than 98%) and co-infection (more than 80%) were found among IS samples from 354 hospitalised infants and children with respiratory illness in this study. Of 273 CAP samples tested, CMV (91.6%), HHV-6 (50.9%), RSV (37.4%), EBV (35.5%), HBoV (28.2%), HHV-7 (18.3%) and rhinovirus (17.2%) were the most commonly detected viruses. Of 81 non- CAP samples tested, CMV (63%), RSV (49.4%), HHV-6 (42%), EBV (24.7%), HHV-7 (13.6%) and HBoV (8.6%) were the dominant viruses detected. The prevalence of several viral agents (rhinovirus, bocavirus, adenovirus and CMV) among IS samples of CAP were significantly higher than that of non-CAP control group. We also found the prevalence of RSV coinfection with HHVs was also higher among CAP group than that of non-CAP control.

Conclusions

With sensitive molecular detection techniques and IS samples, high rates of viral identification were achieved in infants and young children with respiratory illness in a rural area of China. The clinical significance of rhinovirus, bocavirus, adenovirus and HHV (especially CMV) infections should receive greater attention in future treatment and prevention studies of CAP in infants and children.     

连续5 a临床常见非发酵菌分离率变化及耐药性分析

对中国医科大学附属盛京医院2004～2008年5a间非发酵菌的分离情况及其耐药性进行比较和分析,旨在为临床制定合理有效的抗感染治疗方案提供依据。采用琼脂扩散法药敏试验,测定5a间临床常见非发酵菌菌株的药敏,数据统计分析用WHONET5．4软件进行。5a间分离的非发酵菌在革兰阴性杆菌中的比率呈上升趋势,到2008年占革兰阴性杆菌的46．49％。非发酵菌的耐药率逐年升高,其中耐药最严重的是鲍曼不动杆菌。非发酵菌为医院感染的主要致病菌,且对抗菌药物呈多重耐药,临床医生应注重本地区、本治疗区的细菌耐药监测情况,合理选择抗生素,减少耐药菌出现的机会,采取有效措施减少医院内感染的发生和耐药菌在医院内的传播。     

Characterization of Klebsiella Isolates from Natural Receiving Waters and Comparison with Human Isolates

Two hundred sixty-six strains of Klebsiella pneumoniae isolated from natural water sources in geographically diverse areas (Florida, Massachusetts, and Oregon) were analyzed to determine the serotype, biochemical, virulence, and antimicrobial susceptibility differences between these natural strains and human Klebsiella isolates. Sixty of 72 defined serotypes were found among 210 typable strains. Geographic patterns were present, but in general were not pronounced among serotypes. Reactions with 28 biochemical tests showed percentage responses which were very similar to the summaries of primarily human Klebsiella isolates (as reported by Edwards and Ewing, 1972) and that represented diverse geographic sampling. Virulence studies in representative strains showed no geographic variability and little difference from comparable hospital patient-obtained isolates. In contrast to human hospital isolates, strains demonstrated 90% or greater susceptibility to all antibiotics except ampicillin and carbenicillin; and in further contrast, there was little multiple antibiotic resistance beyond that with ampicillin and carbenicillin.     

Complete Genome Sequence of a Novel Human Enterovirus C (HEV-C117) Identified in a Child with Community-Acquired Pneumonia

The new enterovirus C-117 strain belongs to the human enterovirus C species in the Picornaviridae family. We describe the characterization of the complete genome of this strain identified in a respiratory specimen of a child enrolled in the Community-Acquired Pneumonia Pediatric Research Initiative (CAP-PRI) study evaluating the etiology of community-acquired pneumonia (CAP).     

Predictors and Outcome of Hypoxemia in Severely Malnourished Children under Five with Pneumonia: A Case Control Design

Background

There is lack of information in the medical literature on predictors of hypoxemia in severely malnourished children with pneumonia, although hypoxemia is common and is often associated with fatal outcome in this population. We explored the predictors of hypoxemia in under-five children who were hospitalized for the management of pneumonia and severe acute malnutrition (SAM).

Methods

In this unmatched case-control design, SAM children of both sexes, aged 0–59 months, admitted to the Dhaka Hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) with radiological pneumonia and hypoxemia during April 2011 to April 2012 were studied. SAM children with pneumonia and hypoxemia (SpO₂<90%) constituted the cases (n = 37), and randomly selected SAM children with pneumonia but without hypoxemia constituted controls (n = 111).

Results

The case-fatality was significantly higher among the cases than the controls (30% vs. 4%; p<0.001). In logistic regression analysis, after adjusting for potential confounders such as nasal flaring, head nodding, inability to drink, and crackles in lungs, fast breathing (95% CI = 1.09–13.55), lower chest wall in-drawing (95% CI = 2.48–43.41), and convulsion at admission (95% CI = 3.14–234.01) were identified as independent predictors of hypoxemia in this population. The sensitivity of fast breathing, lower chest wall in-drawing and convulsion at admission and their 95% confidence intervals (CI) to predict hypoxemia were 84 (67–93)%, 89 (74–96)%, and 19 (9–36)% respectively, and their specificity were 53 (43–63)%, 60 (51–69)% and 98 (93–100)% respectively.

Conclusion and Significance

Fast breathing and lower chest wall in-drawing were the best predictors of hypoxemia in SAM children with pneumonia. There thus, in resources poor settings where pulse oximetry is not available, identification of these simple clinical predictors of hypoxemia in such children could be reliably used for early O₂ supplementation in addition to other appropriate management to reduce morbidity and deaths.     

Analysis of Human Blood Plasma Proteome from Ten Healthy Volunteers from Indian Population

Analysis of any mammalian plasma proteome is a challenge, particularly by mass spectrometry, due to the presence of albumin and other abundant proteins which can mask the detection of low abundant proteins. As detection of human plasma proteins is valuable in diagnostics, exploring various workflows with minimal fractionation prior to mass spectral analysis, is required in order to study population diversity involving analysis in a large cohort of samples. Here, we used ‘reference plasma sample’, a pool of plasma from 10 healthy individuals from Indian population in the age group of 25–60 yrs including 5 males and 5 females. The 14 abundant proteins were immunodepleted from plasma and then evaluated by three different workflows for proteome analysis using a nanoflow reverse phase liquid chromatography system coupled to a LTQ Orbitrap Velos mass spectrometer. The analysis of reference plasma sample a) without prefractionation, b) after prefractionation at peptide level by strong cation exchange chromatography and c) after prefractionation at protein level by sodium dodecyl sulfate polyacrylamide gel electrophoresis, led to the identification of 194, 251 and 342 proteins respectively. Together, a comprehensive dataset of 517 unique proteins was achieved from all the three workflows, including 271 proteins with high confidence identified by≥2 unique peptides in any of the workflows or identified by single peptide in any of the two workflows. A total of 70 proteins were common in all the three workflows. Some of the proteins were unique to our study and could be specific to Indian population. The high-confidence dataset obtained from our study may be useful for studying the population diversity, in discovery and validation process for biomarker identification.     

Phylogenetic Analysis of Isolates of Pasteurella pneumotropica from Laboratory Animals Based on the gyrB Gene Sequence.

Phylogenetic analysis using the gyrB sequence was performed to investigate the genetic relevance among 49 isolates of P. pneumotropica. In the phylogeny, the isolates were clearly classified into three groups as follows: group A for the isolates of biotype Jawetz derived from mice, group B for the isolates of biotype Jawetz derived from rats, and group C for the isolates of biotype Heyl. These results suggest that the gyrB sequence of P. pneumotropica differs between the isolates of two biotypes, and also between the isolates derived from mice and rats in the biotype Jawetz.     

Molecular Typing of CTX-M-Producing Escherichia coli Isolates from Environmental Water,Swine Feces,Specimens from Healthy Humans,and Human Patients

CTX-M-producing Escherichia coli is the predominant type of extended-spectrum β-lactamase (ESBL)-producing E. coli worldwide. In this study, molecular typing was conducted for 139 CTX-M-producing E. coli isolates, phenotypically positive for ESBLs, isolated from environmental water, swine, healthy humans, and hospitalized patients in Hangzhou, China. The antibiotic resistance profiles of the isolates for the cephalosporins and fluoroquinolones were determined. The isolates showed 100% resistance to cefotaxime and ceftriaxone while maintaining relatively high susceptibility to cefoxitin, cefepime, and ceftazidime. A total of 61.9% (86/139) of the isolates, regardless of origin, showed high resistance to fluoroquinolones. PCRs and DNA sequencing indicated that bla_CTX-M-14 was the most prevalent CTX-M-9 group gene and that bla_CTX-M-15 and bla_CTX-M-55 were the dominant CTX-M-1 group genes. Isolates from all sources with CTX-M types belonging to the CTX-M-1 or CTX-M-9 group were most frequently associated with epidemics. Molecular homology analysis of the isolates, conducted by phylogenetic grouping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST), demonstrated that the dominant clones belonged to B2-ST131, D-ST648, D-ST38, or A-CC10. These four sequence types (STs) were discovered in E. coli isolates both from humans and from environmental water, suggesting frequent and continuous intercompartment transmission between humans and the aquatic environment. Seven novel sequence types were identified in the current study. In conclusion, this study is the first to report the molecular homology analysis of CTX-M-producing E. coli isolates collected from water, swine, and healthy and hospitalized humans, suggesting that pathogens in the environment might originate both from humans and from animals.     

Cloning and Phylogenetic Analysis of Coat Protein of Barley yellow dwarf virus Isolates from Different Regions of Pakistan

Barley yellow dwarf virus (BYDVs) is an emerging threat for wheat and may seriously threaten its production, especially as climate change may result in increased infestation by aphids, the insect vectors of the virus. To assess the possibility of using pathogen‐derived resistance against the virus, the genetic diversity of BYDVs originating from different wheat‐growing areas of Pakistan where its incidence has been higher was investigated. Wheat samples with suspected symptoms of BYDVs were screened for the presence of Barley yellow dwarf and Cereal yellow dwarf viruses (B/CYDVs) subgroup 1 (Barley yellow dwarf virus‐PAV, BYDV‐MAV, BYDV‐SGV) and subgroup II (BYDV‐RPV, CYDV‐RPV, BYDV‐GPV) by PCR using basic multiplex oligonucleotides designed on coat protein (CP) of the virus. Of 37 samples tested, 13 were positive for BYDV subgroup I and only one sample was positive for BYDV subgroup II. Samples positive for subgroup I were further tested by PCR, and results showed that 10 samples were positive for BYDV‐PAV and three for BYDV‐MAV. DNA sequences of CP region of nine isolates (BYDV‐PAV) were determined and compared with available sequences in databases. Sequence analysis showed that three isolates (from Fatehjang, Nowshera and Attock districts) had maximum identity (92.8–94.6%) to BYDV‐PAS, and six isolates (from Peshawar, Islamabad Swabi and Faisalabad districts) had maximum identity (99.3–99.7%) to BYDV‐PAV. Thus BYDV‐PAV species may be dominant in northern wheat‐growing areas of Pakistan. The conserved nature of the BYDVs suggests that pathogen‐derived resistance strategies targeting the coat protein of the virus are likely to provide protection under field conditions.     

Molecular Epidemiology and Phylogenetic Analysis of Human Adenovirus Caused an Outbreak in Taiwan during 2011

An outbreak of adenovirus has been surveyed in Taiwan in 2011. To better understand the evolution and epidemiology of adenovirus in Taiwan, full-length sequence of hexon and fiber coapsid protein was analyzed using series of phylogenetic and dynamic evolution tools. Six different serotypes were identified in this outbreak and the species B was predominant (HAdV-3, 71.50%; HAdV-7, 15.46%). The most frequent diagnosis was acute tonsillitis (54.59%) and bronchitis (47.83%). Phylogenetic analysis revealed that hexon protein gene sequences were highly conserved for HAdV-3 and HAdV-7 circulation in Taiwan. However, comparison of restriction fragment length polymorphism (RFLP) analysis and phylogenetic trees of fiber gene in HAdV-7 clearly indicated that the predominant genotype in Taiwan has shifted from 7b to 7d. Several positive selection sites were observed in hexon protein. The estimated nucleotide substitution rates of hexon protein of HAdV-3 and HAdV-7 were 0.234×10^-3 substitutions/site/year (95% HPD: 0.387~0.095×10^-3) and 1.107×10^-3 (95% HPD: 0. 541~1.604) respectively; those of the fiber protein of HAdV-3 and HAdV-7 were 1.085×10^-3 (95% HPD: 1.767~0.486) and 0.132×10^-3 (95% HPD: 0.283~0.014) respectively. Phylodynamic analysis by Bayesian skyline plot (BSP) suggested that using individual gene to evaluate the effective population size might possibly cause miscalculation. In summary, the virus evolution is ongoing, and continuous surveillance of this virus evolution will contribute to the control of the epidemic.     

High-Throughput Quantitative Analysis of the Human Intestinal Microbiota with a Phylogenetic Microarray

Gut microbiota carry out key functions in health and participate in the pathogenesis of a growing number of diseases. The aim of this study was to develop a custom microarray that is able to identify hundreds of intestinal bacterial species. We used the Entrez nucleotide database to compile a data set of bacterial 16S rRNA gene sequences isolated from human intestinal and fecal samples. Identified sequences were clustered into separate phylospecies groups. Representative sequences from each phylospecies were used to develop a microbiota microarray based on the Affymetrix GeneChip platform. The designed microbiota array contains probes to 775 different bacterial phylospecies. In our validation experiments, the array correctly identified genomic DNA from all 15 bacterial species used. Microbiota array has a detection sensitivity of at least 1 pg of genomic DNA and can detect bacteria present at a 0.00025% level of overall sample. Using the developed microarray, fecal samples from two healthy children and two healthy adults were analyzed for bacterial presence. Between 227 and 232 species were detected in fecal samples from children, whereas 191 to 208 species were found in adult stools. The majority of identified phylospecies belonged to the classes Clostridia and Bacteroidetes. The microarray revealed putative differences between the gut microbiota of healthy children and adults: fecal samples from adults had more Clostridia and less Bacteroidetes and Proteobacteria than those from children. A number of other putative differences were found at the genus level.In the healthy adult, there are 10¹¹ to 10¹⁴ bacteria colonizing the intestine. This outnumbers the total tissue cells in the body by at least an order of magnitude. The composition and activity of this complex microbial system (called microbiota or microflora) have a major influence on health and disease (9). Commensal microbiota contribute to the trophic functions of the gut (producing fermentation products and vitamins that can be used by intestinal epithelial cells), stimulate the immune function of the gastrointestinal tract, transform or excrete toxic substances, protect the host against invasion by pathogenic species, and modulate gut motility (28, 36). At the same time, recent research incriminates a dysfunctional cross-talk between the host and the microbiota in the pathogenesis of a growing number of disorders, such as irritable bowel syndrome, inflammatory bowel disease, allergic diseases, and gastrointestinal cancer (28).While the intestine in a newborn contains no microbes, immediately after birth the intestine of the infant is colonized by enterobacteria and enterococci. Gradual changes in microbiota composition occur during childhood, with a general reduction in the number of aerobes and facultative anaerobes and an increase in the populations of obligate anaerobic species (27). It is considered that by 2 years of age the microbiota resembles that of an adult, which is dominated in health and disease by species from only four phyla, Firmicutes (predominantly Clostridia; 50 to 70% total bacterial numbers), Bacteroidetes (10 to 30%), Proteobacteria (up to 10%), and Actinobacteria (up to 5%), with 90% believed to be obligate anaerobes (4, 10, 11, 22).Traditionally, microorganisms were detected in intestinal samples and feces by microscopic, biochemical, or physiological methods, or by culturing on selective nutrient media. However, since most intestinal microbiota species are obligate anaerobes, their isolation and culturing are difficult (21, 38, 42). In recent years, new methods based on the use of microarray technology have been utilized for the characterization of complex microbial communities (18, 32, 41, 43, 47). Microarrays represent an excellent choice for the high-throughput analysis of bacterial populations, because many different probes can be placed on one slide or synthesized on one chip, and samples thus can be tested for the presence of many different species simultaneously. Environmental and clinical samples can be interrogated directly, circumventing any need for culturing, and thus nonculturable species can be reliably detected.Several types of microarrays have been used to date to characterize the composition of microbial communities (47). Community genome arrays are constructed using whole genomic DNA (gDNA) isolated from pure culture strains (46). Functional gene arrays contain genes encoding key enzymes that are involved in various biochemical processes, and they are useful for monitoring physiological changes in microbial communities (14, 45). Phylogenetic oligonucleotide arrays contain probes derived from rRNA sequence information and are ideally suited for the analysis of microbial community composition structure and variance. Different types of phylogenetic arrays have been designed for these purposes (26, 30, 31).A number of projects performed in the last several years focused on sampling the diversity of human microbiota by the cloning and subsequent sequencing of the 16S rRNA genes isolated from gastrointestinal and fecal samples (5, 10, 13, 23, 38). In this project, we have designed, developed, and validated a custom microbiota microarray containing 16S rRNA genes probes to 775 different microbial phylospecies of human intestinal bacteria. We also have tested the applicability of this array to profiling the microbiota populations in fecal samples isolated from two adult and two child volunteers.     

Assessment of an Enterovirus Sewage Surveillance System by Comparison of Clinical Isolates with Sewage Isolates from Milwaukee, Wisconsin, Collected August 1994 to December 2002

The quantity and serotypes of enteroviruses (EVs) in the influent of a local sewage treatment plant were compared to local clinical EV cases to determine if testing of sewage is adequate for an EV surveillance system. The study was carried out from August 1994 to December 2002. Monthly influent specimens were processed by organic flocculation, and dilutions of concentrate were inoculated onto a number of different cell types for virus isolation. EVs were detected in 88 of 100 monthly influent samples. Sewage EV titers were calculated by using software provided by the U.S. Environmental Protection Agency for most-probable-number determination. All 1,068 sewage EV isolates were further grouped (echovirus, coxsackievirus B, coxsackievirus A, or poliovirus) by cell culture host range analysis (growth pattern of isolates on passage to seven cell lines), and 39.0% of the 1,022 EV isolates categorized as non-poliovirus EVs were specifically serotyped. For clinical cases, primary virus isolation tests were performed on specimens submitted by local hospitals and EV isolates submitted by hospitals were serotyped. Clinical EVs were documented for 81 of the 100 months studied. In all, 694 EV isolates from clinical cases were serotyped. Annually, between 4 and 11 different serotypes of non-poliovirus EVs were identified in sewage and from 9 to 19 different non-poliovirus EV serotypes were identified from clinical specimens. Usually, the most commonly detected sewage EV serotypes were similar to the most commonly detected clinical serotypes; e.g., for 1997, echovirus 6 accounted for 53.1% of the typed sewage isolates and 39.4% of the clinical infections, while in 1998, echovirus 30 accounted for 50.0 and 46.1%, respectively. In 1999, 60.3% of the EVs from clinical cases and 79.7% of the sewage isolates were echovirus 11; in 2000, 33.3% of the EVs from clinical cases and 40.7% of the sewage isolates were coxsackievirus B5; and in 2001, 44.1% of the EVs from clinical cases and 36.2% of the sewage isolates were echovirus 13. Annual peaks of both sewage EV titers and clinical cases occurred in late summer or early fall. In some years, early spring sewage EVs portended some of the EVs that would predominate clinically during the following summer.