We Have Numbers Of Free Samples

Currently there are no cheap and accurate methods of detection for pneumonia in developing countries. Lower respiratory illness accounted for 85% of deaths in low income economies in 2015 [1].

Engineering Goal

The goal is to engineer a fast and reusable device that can help doctors determine which pathogens are causing pneumonia by measuring dissolved oxygen, hydrogen peroxide, and ferritin concentration in the sputum of patient

Purpose

The purpose of this project is to engineer a device to help doctors accurately identify the three most common pneumonia causing pathogens: Streptococcus pneumoniae, Legionella pneumophila, and Haemophilus influenzae. Each of these bacteria has a unique characteristic that can be used to detect their presence. Streptococcus pneumoniae is known to produce small amounts of Hydrogen peroxide, Legionella pneumophila can only survive in aerobic conditions while the other two are anaerobic bacteria, and Haemophilus influenzae can only survive in the presence of iron/ferritin.

Background

A common issue that doctors face when diagnosing patients with pneumonia is identifying the specific pathogen that is causing the infection. Current methods are able to accurately identify all pathogens present in a patient’s body; however, they are unable to determine the specific pathogen causing pneumonia [2]. There may be other infections occurring simultaneously in the patient’s body. As a result, doctors may prescribe antibiotics to treat a viral pneumonia infection which will have no effect on the patient. To counter this issue, scientists created tests that are specific to a single pathogen. The issue with these tests that arises when detecting pneumonia causing pathogens, is that samples are taken from regions of the body where the pathogens causing pneumonia are not present [2]. For example, a common test for pneumonia is a nasopharyngeal swab which is inaccurate because the pneumonia causing pathogens settle in the lower respiratory tract and not in the nasopharynx. The solution to this problem is to take a sputum sample of patients with pneumonia. Sputum sampling is ideal because it is produced directly in the lower respiratory tract. The main reason it is not a mainstream method of sampling is because it can be difficult for patients to produce sputum spontaneously. However, by using the fact that patients already produce excess sputum during a pneumonia infection and using the existing protocol for inducing sputum, sputum can become a feasible method of sampling.

Procedure

Procedure for Oxygen Gas Generation:

1) Skin one potato

2) Cut the potato into small cubes to maximize surface area

3) Put the potatoes into an empty water bottle

4) Take the cap of this water bottle and drill a quarter inch hole in the center

5) Place quarter inch acrylic tubing 6 inches into the hole and seal the edges of the hole

6) Measure 1 cup of 3% Hydrogen Peroxide

7) Put the potatoes and Hydrogen Peroxide into the cup and tighten the cap.

Artificial Sputum Production:

1) Boil ½ cup of water

2) Add three packets of gelatin and use a spoon to soak all of the powder into the water

3) Once all the gelatin is submerged in the water, mix gently for 30 seconds then let the solution sit for 2 minutes

4) Add ½ cup corn syrup and mix immediately

5) Drain the excess water to isolate the Artificial Sputum

Materials

1) Unflavored gelatin

2) Karo Corn Syrup

3) Boiled water

4) 3% Hydrogen Peroxide

5) Skinned and cubed potatoes

6) Hydrogen Peroxide test strip

7) Plastic water bottle for oxygen gas production apparatus

8) ¼ inch diameter Vinyl tubing for transport of oxygen gas

9) ½ inch and 1-inch measuring cups

10)  Pipette capable of accurately measuring to 23 ?L

11)  Pipette tips

12)  10 test tubes (capable of measuring to 2 mL)

Results

Results from Oxygen Gas Generation:

The preliminary experimentation demonstrated that this apparatus can produce oxygen gas. A characteristic of Hydrogen Peroxide is that it splits into hydrogen and oxygen when reacting with certain enzymes. One such enzyme is named catalase which can be found in potatoes. Evidence of a gas being produced existed after just four minutes of adding the Hydrogen Peroxide. The pressure inside the bottle increased, evident from the inability to compress the plastic bottle. Additionally, bubbles were created which is another sign of a gas being produced in a chemical reaction. Oxygen gas must have been the gas produced because the only other substance capable of becoming a gas in the bottle is hydrogen. However, the hydrogen atoms settled in the bottom of the bottle, forming water, and were not released. The gas produced could not have been carbon dioxide produced by an organism because the water was not drunken from and the bottle was sterilized before use.

Results from Artificial Sputum Production:

The Artificial Sputum produced with gelatin and corn syrup was a success. The substance created had a similar viscosity to sputum produced by humans. This is the only characteristic of human sputum that I attempted to replicate for the Artificial Sputum.

Results

Data Analysis

The results of the Hydrogen Peroxide test strip experiment show that the sensor used for Hydrogen Peroxide identification is a valid method for detecting Hydrogen Peroxide in sputum samples. The sensors were able to detect extremely low quantities of Hydrogen Peroxide. The amounts detected were equivalent to the amount of Hydrogen Peroxide that one Streptococcus pnuemoniae bacteria produces. In true sputum samples collected from patients with pneumonia, there will be many more Streptococcus pnuemoniae bacteria present in the sample, meaning much more Hydrogen Peroxide will be present in the sample. Because the sensors are able to detect the small amount of Hydrogen Peroxide produced by one bacterium, the sensors will also be able detect the greater amount of Hydrogen Peroxide that is found in sputum samples of infected patients.

Design Criteria

The target population for this disease is civilians in developing countries with limited access to hospitals.

Criteria for the device are:

• Low cost (less than $100)
• Simple to use (simple procedure and results easy to interpret)
• Safe (no toxic chemicals)
• Durable
• Fast results (5 hours maximum)
• Very accurate

Future Work

Verification of the ferritin sensors and the dissolved oxygen sensors must still be completed. For verification of the dissolved oxygen sensor, I will use the oxygen generation apparatus to oxygenate the sputum sample. I will then use the dissolved oxygen sensor to measure the concentration of oxygen in the sputum sample and compare this to the true value. Additionally, the amount of oxygen inputted into the sample must be in the range of 6-6.7 mg/L because this is the range that the bacteria Legionella pneumophila survives in [3]. This same method of verification will be used to verify the ferritin sensor except with the ferritin protein instead of oxygen. Another future step to complete is the use of professionally produced Artificial Sputum. The sensors will have to be verified again using this sputum is used; however, this professional sputum may produce more accurate results.

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