A sample of an unknown bacterium was collected from a patient suffering with well documented symptoms. In order to proceed with any method of treatment, the bacterium causing the infection needs to be classified. The intent of the experiment was to identify the bacterium from a DNA sample through precise laboratory procedures. The culture grown bacterial cells were put into a centrifuge tube in order to isolate the bacterial DNA needed for PCR amplification. By using PCR the sample was copied and produced many reliable DNA sequences. Microconcentrator column was applied to purify the PCR products. The second last step of the procedure was to sequence explicit small subunit ribosomal RNA (16S rRNA) parts of the bacterium, by producing many separate copies of the DNA and then linking them together. After the sequences have been linked, an entire bacterial DNA was ready for identification by comparison with other 16S rRNA sequences using BLAST search tool. The experiment successfully identified Bartonella henselae as the agent of infection. The sample was extracted from the lymph node of a patient infected with a Cat scratch disease.
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Ridder GJ, Boedeker CC, Technau-Ihling K, Grunow R, Sander A et al. (2002). Role of Cat-Scratch Disease in Lymphadenopathy in the Head and Neck. Clin Infect Dis 35: 643-649
Cat scratch disease (CSD) is caused by a bacterium called Bartonella henselae. The foremost way to pass the infection to an individual is through a close contact with a cat, kittens in particular. By being bitten or scratched, the disease causes an enlargement of lymph nodes, mostly around the neck and head area. Disease known by the name Lymphadenopathy is associated with the lymph node swellings. There are many symptoms of the disease, from uncomplicated fever and headache to adynamia and skin rash. The study tries to link CSD infection with patients with unknown cervical enlargements. The purpose is to improve the ability to detect Cat scratch disease, a pathogen with an increasing number of victims.
The subjects in the study were 454 chosen patients with head enlargements of unknown origin. Ultrasonography and Magnetic Resonance Imaging were assigned for each patient to take a deeper look into the infection. For CSD to be detected three factors have to be identified: clinical symptoms assigned for CSD; presence of antibodies against B. henselae bacterium; and presence of bacterial DNA within the lymph nodes. PCR procedures were used to extract and amplify DNA from enlarged lymph nodes of the patients. Secondly, each patient was tested for B. henselae antibodies, by taking serum samples and running an immunofluorescence antibody test. The results have been shown in a table of Bartonella positive or negative patients.
The outcome of the experiment was that 156 (34.4%) of the 454 patients, who had enlarged lymph nodes, came out to have some kind of an infectious disease. Out of these numbers, 61 (13.4%) of the patients have been identified with all three CSD symptoms. There was no major difference in infections between men and women. In addition, the age for infected individuals was not specified to a certain age group, the scope of cases was from as young as 4 to 89. The results of the experiment will be cited within my discussion to go into a more extensive detail showing how B. henselae is a disease that needs more clinical attention. Secondly, the article goes into deep detail of the Cat scratch disease therefore there is a deep connection between my scientific article and the disease which is caused by the bacterium identified in my laboratory. To explore the way the disease can be transmitted and to underline the importance of PCR techniques for disease identification I will use the CSD article in my experiment to get a better understanding view of the disease itself.
Annotation # 2
Maertzdorf J, Wang CK, Brown JB, Quinto JD, Chu M, de Graaf M et al. (2004). Real- Time Reverse Transcriptase PCR Assay for Detection of Human Metapneumoviruses from All Known Genetic Lineages. J Clin Microbiol 42: 981-986
The objective of the laboratory was to investigate the disease Canine ehrlichiosis which is mainly caused by a parasite. The disease is an infection related specifically to a member of the Canidae family, a dog. Early detection is critical to treat Canine ehrlichiosis successfully. In order to detect the disease within the organism's cells, a fast, reliable and highly sensitive method is needed. Immunofluorescent antibody test (IFA) is the most widely used technique for infectious detection and it usually takes about four weeks to obtain the results. The problem with the indentifying Canine ehrlichiosis is that it takes about three weeks for the antibodies to form. This laboratory was done to find the most effective method in order to eliminate this timing issue. Polymerase chain reaction (PCR) linked with the chemiluminescent hybridization (CH) were the primary techniques that proved to be most useful. PCR/CH results were compared to another test, named cell culture isolation (CCI). The experiment specifically attempted to prove which one of the tests happens to be superior.
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Ehrlichia canis is the parasite that causes infection, and the 16S rRNA gene sequence of this organism was run through PCR/CH. Nine healthy male Beagles were used as the subjects of the study. Each dog, clinically checked prior to experiment, was injected with culture grown E. canis cells. All nine subjects were separated into three groups of diverse (lowest to highest) concentrations of the infectious dosage. At certain time intervals the objects of the study were tested for any signs of the infection. Twenty oligonucleotides were selected from the 16S rRNA gene in order to detect E. canis. The PCR amplification consisted of two stages; first, starting with three cycles of denaturation (95°C), annealing and extension (both at 50°C) and the second stage followed the same process, temperature and order of methods, the only difference was a use of forty repetitive cycles. CH was used to confirm the identity of products.
Results of the study showed that six out of nine dogs, after an injection with E.canis cells, were infected. PCR/CH method identified the antibodies for the disease a week earlier than cell culture isolation. CCI showed equally high sensitivity in the E.canis recognition but it took much longer to get the results. On the other hand, PCR products were not easily seen through gel electrophoresis, but they were detected with much higher quality when tested under CH. The experiment had proven that polymerase chain reaction linked with chemiluminescent hybridization were, together, highly advantageous. The information from the article will be cited within my experiment in order to emphasize the intensity and capabilities of PCR testing in clinical infection detections.
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