OBJECTIVEBacterial cultures from respiratory aspirate or sputum have been the conventional diagnostic method for pneumonia, but the results of culture was often affected by early extensive use of antibiotics, sample collection and delivery. The objective of this study was to explore application of the combined detection of culture and polymerase chain reaction (PCR) assay in hospitalized children with pneumonia.

METHODSTotally 187 hospitalized children with pneumonia were enrolled. The age of the patients ranged from 1 month to 10 years, 124 were male, 63 female; 175 of the patients received antibiotics treatment before admission. Deep respiratory aspirate sample from patients was cultured by Streptococcus pneumoniae selective plate, Hemophilus influenzae selective plate and conventional plate. The aspirate samples were also amplified for DNA of 14 bacteria with target enriched multiplex polymerase chain reaction (Tem-PCR) and detected with Luminex xMAP technology platform.

RESULTSThe total positive rate by bacterial culture was 40.1% (75/187), of which 17.1% (24/187) were Hemophilus influenzae b, 8.6% (16/187) were Escherichia coli, 6.4% (12/187) were Klebsiella pneumoniae, 4.8% (9/187) were Staphylococcus aureus, 3.7% (7/187) were Streptococcus pneumoniae, 1.6% (3/187) were Pseudomonas aeruginosa, 1.1% (2/187) were Acinetobacter baumannii, and 1.1% (2/187) were Enterobacter cloacae. The total positive rate by combined detection of culture and Tem-PCR assay were 78.6% (147/187), of which 28.9% (54/187) were Hemophilus influenzae b, 19.3% (36/187) were Streptococcus pneumoniae, 8.6% (16/187) were Escherichia coli, 6.4% (12/187) were Klebsiella pneumoniae, 5.9% (11/187) were Staphylococcus aureus, 5.9% (11/187) were Acinetobacter baumannii, 2.7% (5/187) were Pseudomonas aeruginosa, and 1.1% (2/187) were Enterobacter cloacae.

CONCLUSIONThe Tem-PCR assay may increase the detection rate of Hemophilus influenzae b, Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii. The Combined detection may increase the positive rate of bacterial pathogens in hospitalized children with pneumonia, and the results might reflect the real patterns of bacterial etiology. The Tem-PCR needs further improvement for diagnosis of Escherichia coli and Klebsiella pneumoniae.

Child ; Child, Preschool ; Colony Count, Microbial ; Female ; Haemophilus influenzae ; genetics ; isolation & purification ; Humans ; Infant ; Male ; Pneumonia, Bacterial ; microbiology ; Polymerase Chain Reaction ; Streptococcus pneumoniae ; genetics ; isolation & purification

OBJECTIVETo establish multiplex PCR-based assays for detecting H.influenzae and H.parainfluenzae. And the PCR-based assays were applied to detect the carriage rates of H.influenzae and H.parainfluenzae in nasopharyngeal swab specimens which were collected from healthy children.

METHODSMultiplex primers for species-specific PCR were designed by using DNAstar soft based on the sequences of 16S rRNA genes from genus Haemophilus to detect H.influenzae and H.parainfluenzae.

RESULTSThe sensitivity of the 16S rRNA PCR assay for detecting H.influenzae and H.parainfluenzae was 97.53% and 100% respectively, and the specificity was 95.89% and 96.63% respectively. Youden's Index on the ability to detect H.influenzae and H.parainfluenzae was 0.9342 and 0.9663 respectively. 666 nasopharyngeal swab specimens were collected from healthy children. The detection rates of H.influenzae and H.parainfluenzae were 14.11% and 16.07% respectively by using isolation and culture methods. The detection rates of H.influenzae and H.parainfluenzae were 43.54% and 57.96% respectively by 16S rRNA PCR assays. The carriage rates of serotypes a, b, c, d, e, f and non-typeable isolates were 0% (0/666), 0.15% (1/666), 1.20% (8/666), 0.15% (1/666), 1.20% (8/666), 1.80% (12/666), 95.50% (636/666) respectively.

CONCLUSIONThe multiplex PCR assays were very rapid, reliable and feasible methods for detection of H.influenzae and H.parainfluenzae in pharyngeal swab specimens which were compared to conventional isolation and culture methods. 95.5% of H.influenzae strains in healthy children were nontypeable. The encapsulated or typable strains were mainly three serotypes which was c, e, and f serotype.

Haemophilus influenzae ; classification ; genetics ; isolation & purification ; Haemophilus parainfluenzae ; classification ; genetics ; isolation & purification ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Nasopharynx ; microbiology ; RNA, Bacterial ; genetics ; RNA, Ribosomal, 16S ; genetics ; Sensitivity and Specificity

Child ; China ; Community-Acquired Infections ; microbiology ; Drug Resistance, Bacterial ; genetics ; Haemophilus influenzae type b ; drug effects ; genetics ; isolation & purification ; Humans ; Microbial Sensitivity Tests ; Pneumonia ; microbiology

OBJECTIVETo set up a rapid and simple method for identificating bacteria by 16S-23SrDNA intergenic spacer regions (ISRs).

METHODSPolymorphic products of PCR from ISRs were selected on agarose gel and sequenced directly using purified fragments by excising the gel without cloning. Nucleotide sequences were compared with GenBank databases and analyzed by DNAMAN program.

RESULTSThere was only a single product in streptococcus genus after PCR amplification of 16S-23SrDNA ISRs. Five streptococcal species were obtained from 7 strains of streptococcus. Two major amplicons were consistently generated for 8 strains of Haemophilus influenzae (H. influenzae). The sequence data showed that they all belonged to H. influenzae type b on GenBank databases.

CONCLUSIONPCR and direct sequencing of 16S-23SrDNA ISRs were very successful methods for bacterial species identification.

Base Sequence ; DNA, Ribosomal Spacer ; chemistry ; Haemophilus influenzae ; genetics ; isolation & purification ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Streptococcus ; genetics ; isolation & purification

This study was performed to investigate polymerase chain reaction-based detection of bacterial DNA in middle ear fluid and assess the correlation between the PCR-positive rate with several factors associated with middle ear effusion. The purpose was to gain a further understanding of bacterial infection as a major cause of otitis media with effusion. Of the 278 specimens of middle ear fluid, 39 (14%) tested positive by ordinary culture. The overall detection rate of bacterial DNA using the PCR method was 36.7% for middle ear effusion, and bacterial DNA detection rates of Hemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis in the middle ear effusion were 29.1%, 4.7% and 10.8%, respectively. The bacterial DNA detection rate was higher in ears with a history of acute otitis media than those without the history. High detection rates were observed in patients younger than 48 months who have had a higher tendency to present with acute otitis media. We concluded that PCR is a more sensitive method for the detection of bacteria in middle ear effusion than ordinary culture, and acute otitis media is a major contributor to the pathogenesis of otitis media with effusion.
Child ; Child, Preschool ; Chronic Disease ; DNA, Bacterial/analysis ; Haemophilus Infections/*diagnosis ; Haemophilus influenzae/genetics/*isolation & purification ; Humans ; Infant ; Moraxella (Branhamella) catarrhalis/genetics/isolation & purification ; Moraxellaceae Infections/diagnosis ; Otitis Media with Effusion/*diagnosis/*microbiology ; Polymerase Chain Reaction ; Research Support, Non-U.S. Gov't ; Streptococcal Infections/diagnosis ; Streptococcus pneumoniae/genetics/isolation & purification

OBJECTIVEHaemophilus (H.) influenzae is a gram-negative bacillus that is a common commensal organism of the human upper respiratory tract and an important cause of human diseases such as pneumonia, meningitis, septicemia, epiglottitis and cellulitis. Strains of H. influenzae are classified according to their capsular polysaccharide. There are six serotypes, designated as a through f. In addition, there are nonencapsulated strains. Although the type of infectious diseases caused by H. influenzae has changed considerably in recent years because of the widespread and routine immunization of children against type b H. influenzae (Hib), Hib remains an important pathogen. Ampicillin is the drug of choice for treating many infections caused by H. influenzae, but its usefulness has been compromised by the increasing prevalence of ampicillin-resistant strains. The continued monitoring of resistant strains by using genotyping methods may provide insights into the epidemiology of transmission. A molecular epidemiological study of ampicillin-resistant H. influenzae derived from nasopharyngeal swabs specimens of children less than 5 years of age with respiratory tract infection were investigated in this study.

METHODSA total of 899 isolates were collected from Beijing, Shanghai, and Guangzhou during 2000-2003. Susceptibility to ampicillin was determined by using E-test. Ampicillin-resistant H. influenzae strains were selected according to National Committee for Clinical Laboratory Standards (NCCLS) 2002 breakpoints. Nested PCR method with primers specific for bexA gene and b capsulate type-specific gene was established. Genotyping by pulsed-field gel electrophoresis (PFGE) and multiplex PCR assay was performed for all ampicillin-resistant H. influenzae strains.

RESULTSSeventy-four ampicillin-resistant H. influenzae strains were obtained. Two strains were positive by nested PCR, characterized as b genotype. The incidence of Hib in ampicillin-resistant H. influenzae strains was 2.7%; 38 genotypes were detected by PFGE. Detection of five types strains of clonal dissemination by PFGE accounted for 55.4% in all ampicillin-resistant H. influenzae strains. Among them eighteen H. influenzae strains belonged to one type, accounted for 24.3% in all ampicillin-resistant H. influenzae strains. Thirty one genotypes were identified by multiplex PCR assay for ampicillin-resistant H. influenzae. The identity ratio of PFGE and multiplex PCR was 63.5%.

CONCLUSIONIn Beijing, Shanghai and Guangzhou areas 55.4% of ampicillin-resistant H. influenzae strains had clonal dissemination during the 4 years.

Ampicillin Resistance ; genetics ; Anti-Bacterial Agents ; pharmacology ; Child, Preschool ; China ; epidemiology ; DNA, Bacterial ; genetics ; Drug Resistance, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; Genotype ; Haemophilus Infections ; epidemiology ; microbiology ; Haemophilus influenzae ; classification ; genetics ; isolation & purification ; Humans ; Microbial Sensitivity Tests ; Molecular Epidemiology ; Nasopharynx ; microbiology ; Polymerase Chain Reaction ; Respiratory Tract Infections ; microbiology