Rapid Microbial Detection: Decreasing the Number of Deaths Caused by Sepsis

Dina Eloseily

10 January 2018

Intern/Mentor G/T
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On January 5th, 2016, eighteen-year-old Alexa Bernard had gone to an oral surgeon to

remove her wisdom teeth; a common procedure for teenagers. After the surgery, the doctor

assured her that it had been successful and Alexa would recover within a few days. However,

later that night, Alexa had lost consciousness while walking to her room to grab an extra pillow

to prop up her head and stop the blood from running down her throat. Petrified, her mother called

911, and an ambulance came to escort her to the emergency room. Her vitals were dropping and

fluid was accumulating in her lungs; her health was rapidly decaying. The last thing Alexa

remembered was screaming in pain as nurses inserted IVs into her arm. Seven days later, she

woke up in the hospital to find her family from across the country in her hospital room with her.

She learned that she had gone into septic shock when she entered the emergency room, and the

infected blood in her brain had prevented her from being able to remember events of the past

week. Alexa’s body had defeated the sepsis, but she still had months of pain ahead of her.

Doctors concluded that she had been infected during her oral surgery, supposedly by either a

contaminated needle or an expired anesthetic, and within just eleven hours, Alexa had fallen

fatally ill. While Alexa was extremely lucky to survive, millions of other Americans are not as

fortunate in their experiences with sepsis (Bernard).

An unpredictable and highly fatal condition, sepsis is one of the leading causes of death

in hospitals today (NIH). This infection is most often caused by bacteremia, the presence of

bacteria in the blood. There are multiple sources of this condition, ranging from a local infection,

such as a urinary tract infection, to bacteria entering the bloodstream during surgery, as

commonly seen in hospitals. When bacteremia occurs, the immune system works to combat the

toxins by secreting chemicals into the bloodstream; however, if the bacteria persists, blood clots

and defective blood flow may occur. If the case is severe, the patient suffers from organ failure
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and in the most severe cases, the patient goes into septic shock. Dangerously low blood pressure,

weak heart function, and vital organ failure, are all characteristics of septic shock, which can

result in death (“Sepsis”).

Sepsis affects an average of one million Americans each year, and this number has been

increasing. It is possible for anyone to be infected, however it is most commonly found in infants

and young children, the elderly, cancer patients, and individuals who have recently undergone a

medical procedure. Once a patient is diagnosed with sepsis, he or she is given antibiotics, and

often admitted into the intensive care unit. For severe conditions, the patient may require kidney

dialysis, or may even need to be placed on a breathing tube if they are unable to breathe on their

own. Even for those who survive sepsis, there are lasting effects, such as an increased risk of

future infections (NIH).

Blood culture results and symptoms both factor into the diagnosis of bacteremia and

sepsis (NIH). To complete a blood culture, blood is drawn, sent to a laboratory, and analyzed for

bacteria. If bacteria are found, subsequent testing is done to identify the appropriate antibiotics

needed to treat the patient. Because sepsis is so severe, it is essential for treatment to begin as

early as possible, meaning that an early diagnosis is crucial to the patient’s health (Nordenson).

The goal of antibiotic susceptibility testing (AST) is to detect possible drug resistances and

assure susceptibility to drugs for particular infections. As of today, there are only a few approved

automated AST instruments used in the United States; however, these products are time-

consuming, expensive, and may be difficult to use. I am interning at the University of Maryland,

Baltimore County under Research Professor Govind Rao in the Center for Advanced Sensor

Technology (CAST). I am working with Mustafa Al-Adhami on a low-cost device for rapid

microbial detection that requires little to no training for the end user.
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One of the most common commercial products is the BD Phoenix by BD Diagnostics.

This instrument has the ability to simultaneously test ninety-nine panels using turbidimetry and

colorimetry methods. When bacteria are present in a sample, they create turbidity, also known as

haziness, in the solution. Through turbidimetry, this haziness is detected by measuring the loss of

intensity of transmitted light, therefore measuring the presence of microbial particles. Similarly,

colorimetry determines the concentration of a chemical element using a color reagent. By

monitoring the samples every twenty minutes, the Phoenix produces results anywhere between

six to sixteen hours.

A competing product in the market is the Vitek 2 System by BioMérieux; a highly

automated machine that can analyze up to two-hundred-forty simultaneous tests to identify

pathogens responsible for infections, and determine the susceptibility of these bacteria to

antibiotics. This instrument is available in multiple forms that vary in size and automation to

accommodate different demands of laboratories and clinics. Similar to the BD Phoenix, Vitek 2

uses turbidity and colorimetry to identify microorganisms and determine their susceptibility.

Depending on the type of bacteria being tested, Vitek 2 can provide results anywhere between

eight to eighteen hours (Pincus 2-9).

The MicroScan WalkAway is yet another automated instrument that measures

antimicrobial susceptibility. Differing in its method, this system utilizes photometry, the

measurement of light, and fluorogenic identification, the measurement of fluorescence. The

duration and process of analysis is dependent upon the type of bacteria; whether they are gram-

positive or gram-negative (Rhoads et al. 1). The key difference in the two are the thicknesses of

their membranes. Gram-positive bacteria have very thick membranes that can easily absorb

external fluids (Schaalje). Contrastingly, gram-negative bacteria have very thin outer layers that
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are extremely difficult to penetrate, therefore requiring additional time in susceptibility testing:

about five to eighteen hours (Reller et al.).

The final widely-used instrument in the United States is the most time-consuming,

requiring a minimum of twenty-four hours to produce test results. This Sensititre ARIS 2X by

Trek Diagnostics simultaneously analyzes sixty-four test panels, and both gram-negative and

gram-positive bacteria. Similar to the MicroScan WalkAway, the Sensititre measures the change

in fluorescence to detect bacterial growth (Reller et al.).

The device that Mustafa and I are working on has some similar features to the previously

addressed commercial products, while also improving upon some of their major setbacks. This

device uses a photodiode to measure the change in fluorescence of the sample in order to detect

bacteria. Samples are placed into microfluidic acrylic cassettes, then placed into the device

which is just large enough to hold in one’s hand, making it easily portable. Before being placed

into the cassettes, the samples are stained with a blue dye called Resazurin. When this dye comes

in contact with living bacteria, it is reduced to Resorufin, a highly fluorescent purple dye. This

change in fluorescence is quantifiable, which is used to determine the presence of bacteria and

measure its susceptibility to antibiotics (Al-Adhami et al.).

The Oxford Infectious Diseases Society of America highlights that “there is a need for

development of new automated instruments that could provide faster results and also save

money” (Reller et al.). Engineers at CAST, including myself, believe this device is capable of

becoming highly competitive with the other automated AST instruments in the market. The

minimum amount of time required for any of the currently available instruments is five hours;

however, as of now, this device will take no longer than one hour to analyze a sample. Regarding

the costs of these machines, they can add up to anywhere between one to two-hundred thousand
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dollars per year, and our price is expected to be about 10% of that; clearly providing a more cost-

effective alternative (Turner et al.). This product does, however, test just one sample at a time,

while the others are capable of completing many simultaneous tests. This can be attested to the

target audience; this may be used for emergency testing that require rapid results. Along with

that, the device can be easily used without any advanced skills or training necessary, allowing for

facilitated adaptability among hospital staff and a more expedited testing process.

While this device provides many favorable characteristics, it is still a work-in-progress.

Mustafa and I are constantly experimenting working diligently in hopes to have the device

commercially available in the coming months. We are still working towards analyzing blood

samples; a task that has not yet been fully completed. While we still have many obstacles to

overcome, we are working assiduously to facilitate and expedite antibiotic susceptibility testing

to compete with the other currently-available products in the market.

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