Molecular Tests In The Clinical Diagnostics Of Bordetella Infections

Older children and adults often present with mild or atypical pertussis that easily goes unrecognized. In addition, other pathogens[18] can cause pertussis-like illnesses. These factors complicate the clinical diagnosis of pertussis and make the laboratory diagnosis a vital component of effective strategies against the disease in the community. Laboratory diagnosis requires isolation of B. pertussis or B. parapertussis from respiratory secretions, demonstration of specific antigens or nucleic acids in the above secretions, or detection of a serological response to infection. Culture and detection of antigens or nucleic acids yield positive results at the early stages of pertussis, whereas serology is usually the only diagnostic method that can be used at later stages, when microbial antigens and nucleic acids have disappeared from the infected host.

Bordetellae are fastidious organisms. Sampling time, sample collection and transportation methods, and culture conditions affect the sensitivity of culture. Furthermore, culture is a slow procedure; it may take up to 3 weeks before culture results are available for the physician. The direct fluorescence antibody test (DFA) uses polyclonal fluorescein-labeled antibodies to B. pertussis and B. parapertussis for the detection of organisms in nasopharyngeal smears.[19] The method is compromised by inadequate sensitivity, specificity, and subjectivity and should be used only in parallel with culture. The diagnostic sensitivity of serology is very much dependent on the timing of sample collection, choice of antigen, and the immunoglobulin class.[1,20] Assessments of IgG and IgA antibodies to PT and filamentous haemagglutinin (FHA) are the most useful assays. Only antibodies to PT are specific for B. pertussis, antibodies to FHA and Prn may be elicited by B. parapertussis and other bacteria.[21] The diagnostic efficacy of serology is rather low in the acute phase of the disease. At the later stages of pertussis, diagnosis is primarily based on serology. Without se-rology, adolescent and adult pertussis cases are largely overlooked, as the patients do not usually contact a doctor until after a long duration of symptoms.

Molecular diagnostic methods such as PCR have been widely applied to the direct detection of the DNA of fastidious pathogenic bacteria from clinical specimens. PCR has proved a rapid, sensitive, and reliable tool for the diagnostics of pertussis.[10] A variety of regions in the B. pertussis genome have been successfully used as targets, including the PT promoter region, repeated insertion sequence IS481, a DNA region upstream from the porin gene and the adenylate cyclase toxin gene. IS1001-based PCR has been applied to the diagnosis of B. parapertussis infections.[10]

The recent application of fluorescence techniques to in vitro nucleic acid amplification allows simultaneous amplification and sequence-specific detection of PCR products using hybridization probes.[22] The advantages of these real-time PCR methods are that PCR amplification can be carried out in less than an hour and no further analysis of the PCR amplicons (such as gel-electrophoresis and Southern blotting) is required. Because no PCR products need to be handled, contamination risk is minimized. Like with conventional PCR, the crucial issue of the use of these real-time PCR methods is finding good amplification targets allowing sensitive and specific detection of a given pathogen. The target gene region should have sufficient sequence variation to allow differentiation between B. pertussis and B. parapertussis and B. holmesii. However, the gene region should be conserved without any variation on the subspecies level in the regions where the probes bind. Further, multiple copies of the target gene region in the genome increase the sensitivity of the assay. Multiple copies of repeated insertion sequences IS481 and IS1001 are present in the genomes of B. pertussis and B. parapertussis and, therefore, IS-based PCR assays are widely used in diagnostic laboratories. The first diagnostic real-time PCR assay developed for simultaneous detection and identification of B. pertussis and B. parapertussis also targeted at IS481 and IS1001.[23] However, IS481 is also present in the genome of B. holmesii, and the major drawback of the IS-based assays is that B. holmesii and B. pertussis infections cannot be differentiated. The major limitation of the other target genes tested so far is that they are present singly or in low copy numbers and their amplification may not allow as good analytical sensitivity as that of IS-based assays.

Currently, rapid real-time PCR assays are gradually replacing conventional PCR assays in clinical diagnostic laboratories. One of the major challenges in pertussis diagnostics is finding new target gene regions for sensitive and specific detection and identification of Bordetellae directly from clinical specimens. However, PCR can probably never fully replace culture, as the collection of clinical isolates for research purposes is an important task for diagnostic laboratories.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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