Bordetella Pertussis Case Study

Bordetella pertussis also commonly known as whooping cough is a very contagious respiratory infection. This infection is caused by a gram negative coccobacillus. This organism damages ciliated epithelium in the nasopharynx and bronchioles. The toxin produced by Bordetella pertussis paralyses the cilia, causes tissue inflammation, and leads to impaired mucus clearance. In serious cases lymphocytosis occurs due to the inhibition of phagocytic functions, leading to a formation of a mucus exudate. The small airways can be compromised due to this exudate and it predisposes patients to cyanosis and pneumonia (Bentley, May 2013). The Bordetella pertussis infection is divided in three stages according to its clinical course. The first stage last approximately
1 to 2 weeks and the most prominent symptoms are progressive cough, rhinorrhea, conjunctiva infection, excessive lacrimation, and a low grade fever. The second or paroxysmal stage is more severe; cough is present with its characteristic whoop. Patients may also present bouts of apnea. This stage could last up to 10 weeks. In the third stage also called convalescent stage, the cough starts to subside. However, the respiratory infections may recur such as rhinovirus or bronchitis. 1 to 3 weeks is the approximate time to finally recover from this last stage (Nirenberg & Plaviskin, 2012). The contagious transmission is a result of poor hygiene and via poor social etiquette.
Droplets containing the B. pertussis bacteria from coughing and sneezing are the main source of infection in young children and infants. The main infection transmission source are their asymptomatic parents or siblings. (Rittle, 2010) Adolescents and adults, with whom immunity diminishes, leads to an increased number of these individuals contracting the whooping cough. The most vulnerable groups are infants and newborns who lack the vaccination or full immunity. Especially at risk for symptoms of infection are children under 1 year of age. (Loeffelholz,
2012) Studies have demonstrated that those individuals who missed at least one dose of the vaccination are at higher risk to acquire the B. pertussis infection than those who received the full five doses. The efficacy of the pertussis vaccination is complete for this first year. It gradually decreases over the next three years, by the fourth year it is 84% effective if full doses were received. The next three years this efficacy continues falling to 50% which is not sufficient to fight the infection. Furthermore, the individuals who contract the Bordetella pertussis infection tend to present atypical manifestations and the infection is frequently underdiagnosed (Rittle, 2010). Despite vaccination availability for Bordatella pertussis, over 27,000 cases were reported in United States in 2012.This average only represents the cases reported but the estimation for cases in the nation is about 800,000 cases to 3.3 million every year according to the CDC. Approximately 300,000 deaths occur annually worldwide caused by Bordetella pertussis infection. (Loeffelholz, 2012) Whooping cough is denominated as a cyclical infection and occurs two to five times per year, usually late summer and autumn. (Bentley, 2013) The laboratory has been improving techniques. Health care protocols such as early patient isolation have been developed to treat this infection. In the laboratory, there are several options to diagnose patients. The most common one is the routine microbiology culture. The usual specimen are tracheal aspirates or sputum. This test has a high specificity up to 100% but the results can take up to 2 weeks to obtain. There are others types of testing such as Direct Fluorescent Antibody, but it is less sensitive and less specific. There is also some serology testing, which is a routine test in most of the labs, but it is not appropriate in early stage of disease. Molecular testing has become essential with a high specificity (98% to 99%) and sensitivity (up to 95%) to diagnose this infection. (Carmon & Spratling, 2010) There are several molecular instruments in the market available for laboratories. I will focus only on a couple for this document due to the vast material. The first instrument is the FilmArray® from Idaho Technology Inc. This instrument targets a total of 20 viruses and bacteria (Including B. pertussis) in a full comprehensive panel at the same time. Its preparation takes about two minutes hands-on, precise measuring or pipetting is not required, and the turnaround time is about an hour. The biggest advantage for the B. pertussis organism tested by FilmArray® is that specifically detects the B. pertussis and does not cross–react with other subtypes of Bordetella species. Once the panel is set up several molecular steps occur. First, the nucleic acid purification occurs, the sample is lysed by agitation, and the liberating the nucleic acids are then captured, washed, and eluted by using magnetic bead technology. Reverse transcription (RT) is subdivided into two steps. Step one is multiplex PCR, the RT coverts viral RNA into cDNA before amplification. At this point the purified nucleic acid is mixed and subsequently thermocylcing is used for multiplex PCR. The second steps consist of diluting the products from the first step and mixing again with fresh PCR reagents containing fluoresce DNA Dye (LC Green® Plus). This solution is then distributed in wells of the arrays containing primers for different assays that target specific sequences from each of the pathogens detected, including B. pertussis. These primers are "nested" to enhance sensitivity and specificity (Idaho Technologies, 2012). On the last step, the method used for analysis is DNA melting analysis. After the second stage of PCR, the temperature is gradually increased and the fluorescence in each well is monitored and examine to generate a melting curve. The temperature (Tm) at which specific product melts is consistent and predictable for the instrument. The FilmArray® software examines data from each well and reports result at the end of the run of the whole panel including Bordetella pertussis. This test is in vitro and only nasopharyngeal swabs can be tested with this instrument. (Idaho Technologies, 2012) The entire run can last an hour compared to a week or two from routine cultures. The second analyzer is RIDA®GENE Bordetella. This method uses real time multiplex PCR and it is in vitro only. This method also differentiates between this B. pertussis and two other species of Bordetella. This guides healthcare givers to a better treatment and the source of the infection. With this product washes and nasopharyngeal swabs can be used. The first step in this procedure is to isolate DNA from the specimen. That is done by adding 200 μl of water (DNAse free) into a tube. The specimens are added or a swab is broken off into the tube. The tube is closed and then vortexed (R-Biopharm AG, 2013). The test consists of completing the amplification of gene fragments specific for the three Bordetella species. The amplified targets are detected by hydrolysis probes; these probes are labeled with one probe at the end with a quencher. The other end is labeled with fluorescent dye (flourophore). In the targets’ presence the probes hybridize to the amplicons. The initial denaturation takes about one minute at 95°C. It takes about 45 cycles. The annealing takes about 10 seconds at 95°C and extension takes about 15 sec at 60°C; these steps occur in the same time (R-Biopharm AG, 2013). During the extension step, the Taq-polymerase breaks the reporter quencher proximity.
This function emits a fluorescent signal which is detected by an optical unit included in the real-time PCR instrument. The bigger the amount of amplicons formed, the stronger the fluorescence signal will increased. This instrument run internal controls at the same time. The analysis is verified by a software included with the instrument. Reports of positive, negative and patient on the report generated by instrument. The RIDA®GENE Bordetella real-time PCR has a detection limit of ≥ 5 DNA copies per reaction for the three species tested. The detection limit depends on the sample DNA extraction and DNA-concentration (R-Biopharm AG, 2013). The RIDA®GENE Bordetella real-time PCR and FilmArray® are only two examples of a numerous instruments used for respiratory infections. The results can be given in about an hour which highly exceeds the expectative to diagnose a relevant infection such as Bordetella pertussis. It is also significant for doctors to recur on their knowledge for patient’s symptoms, medical history, or background to make a complete diagnosis. The molecular testing is also used in a diverse fields, such as oncology or genetics. There is still a lot of research being done in every molecular diagnostic to keep improving each
test. These two molecular instruments can aid the diagnosis of Bordetella pertussis. This will help healthcare workers for a faster response to a critical situation in a quick manner. They also save time for patients’ treatment and recovery. At the long run, these methods can be very cost effective and over all beneficial for the medical field. The rapid paste of the molecular testing in a diverse fields in the medical world are becoming a revolutionary tool that benefit a whole society.