A single-step polymerase chain reaction for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae.

OBJECTIVE: The critically high prevalence of bacterial otitis media worldwide has prompted a proper disease management. While vaccine development for otitis media is promising, the reliable and effective methods for diagnosis of such etiologic agents are of importance. METHODS: We developed a multiplex polymerase chain reaction assay for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae. Five primer pairs targeting genes fumarate reductase (H. influenzae), outer membrane protein B (M. catarrhalis), major autolysin (S. pneumoniae), capsulation-associated BexA protein (all encapsulated H. influenzae) and 16S rRNA were incorporated in this single-step PCR. Validation of the multiplex PCR was also performed on clinical isolates. RESULTS: The developed multiplex PCR was highly specific, enabling the detection of the target pathogens in a specific manner, either individually or as a mixture of all target organisms. The assay was also found to be sensitive with the lowest detection limit of 1 ng of bacterial DNA. When applied to clinical isolates from diverse specimen sources, the multiplex PCR developed in this study correctly identified each microorganism individually or in a combination of two or more target organisms. All results matched with conventional culture identification. In addition, the ability of such assay to differentiate H. influenzae encapsulation from the study clinical isolates was 100%. CONCLUSION: Our multiplex PCR provides a rapid and accurate diagnostic tool for detection of the 4 target organisms. Such assay would serve as a useful tool for clinicians and epidemiologists in their efforts to the proper treatment and disease management caused by these organisms.

Nosocomial spread of class 1 integron-carrying extensively drug-resistant Pseudomonas aeruginosa isolates in a Thai hospital.

Fifty non-duplicate multiresistant isolates of Pseudomonas aeruginosa from a regional hospital in Northern Thailand were investigated for their antimicrobial susceptibility, presence of class 1 integrons and arrangement of gene cassettes as well as their genetic relationships. All but one isolate were classified as extensively drug-resistant P. aeruginosa (XDR-PA). Forty-one isolates (82%) were found to carry class 1 integrons. Amplification of the variable regions of class 1 integrons revealed seven diverse bands ranging in size from 0.7 kb to 7.0 kb. Sequence analysis of class 1 integron variable regions revealed the presence of several gene cassettes associated with resistance to aminoglycosides (aac, aad and aph), including the aac(3)-Ic cassette reported for the first time in Thailand. Gene cassettes encoding resistance to chloramphenicol (cmlA), ß-lactams (bla(PSE), bla(OXA) and bla(VEB)) and rifampicin (arr) were found. The putative small multidrug resistance protein (smr) and an open-reading frame with unknown function (orfD) were also detected. The aadA6-orfD cassette array was the most common integron found in this study. Integron-positive isolates had higher frequencies of antimicrobial resistance than isolates lacking integrons. Pulsed-field gel electrophoresis (PFGE) demonstrated the occurrence of horizontal gene transfer. Interestingly, a large number of XDR-PA isolates carrying identical integrons clearly exhibited the same PFGE pattern, indicating nosocomial spread of these isolates. The presence of XDR-PA carrying class 1 integrons is implicated in the possible spread of drug-resistant organisms, therefore screening for integron-positive P. aeruginosa might be necessary for protection against nosocomial infection.