TB or not TBPCR based Tuberculosis (TB) Detection on a Chip

Lael Wentland, Albert Nguyen, and Minjung Kim

Global Impact of TBMycobacterium tuberculosis- ⅓ of world population infected with TB- latent or active

[1]

● Leading killer for HIV+→ Difficult to diagnose in HIV+ patients

● Can be multi-drug resistant (MDR-TB)● Difficult for young children to give sputum sample● More accurate test → power supply, labratory, skilled professional

Other Factors Complicating TB Diagnosis

[2]

Engineering: Global Health Challenges

Living on Under $10 a Day

● Consider cost for where the diagnostic is needed?

● Who will be doing the test? - > shortage of health workers

○ Majority of work done by individuals with less training

● Where will this diagnostic be done? - higher level hospitals with electricity

● At what stage of the disease will people be tested?- > ideally earlier

[3]

[2]

Method Pros Con

Culture ● the gold standard!● allows genotyping of the

bacteria● susceptibility testing

● prolonged time to result ( > 2 weeks) ● high cost with delayed diagnostics● difficult to obtain patient samples● $30 per test [5]

Acid Fast Stain ● rapid detection ● inadequate sensitivity/specificty

Interferon Gamma -Release Assays (IGRA)

● tests can be done in less than 24 hours

● requires single patient visit

● need fresh blood samples● test cannot be given to patient who has

been recently ( < 4-6 wks) vaccinated● cost $160-290 [6]

Skin Test • Easy to administer ● cost around $55 [6] per test

Traditional PCR PCR

● High sensitivtiy and sensitivity

● Rapid detection time● No transport requirement● Allows detecton from non

invasive species

● Potential contamination● Unable to assess viability● Limited ability for genotype and

susceptibility testing● $ 5-10 [7]

Current Diagnostic Methods for TB

Adapted from [4]

Final Clinical Diagnosis/Method Mycobacterial Culture

PCR

Tuberculosis 19 Positive 10 Positive8 Negative1 Uncertain

14 Positive5 Negative

Non-tuberculous Mycobacterial Infection

6 Negative 3 Positive3 Negative

6 Negative

Nonmycobacterial infection

33 Negative 29 Negative4 Uncertain

33 Negative

Uncertain 2 2 1 Positive1 Negative

Side by Side Comparison of Mycobacterial Culture and PCR

93% specific

Sensitivity = true positives/(true positive + false negative)Specificity = true negatives/(true negative + false positives)

100% sensitive

[8]

Why use microfluidics for PCR?

● Portable● Cost-effective

o does not require trained staff or infrastructure

● species concentration can be regulated in space and time

● quick and uniform heat and mass transfer because of the high surface to volume ratio

● increased speed and automation opportunities that can potentially reduce contamination

[12]

[11][10]

[9]

General Constraints

● Reach and hold 98C

● Material that will be heated by PID controller

● Pass FDA 510(k) inspection

Global Health Constraints

● Low cost

● Able to function in a variety of environments-durable

● Easy to use and minimal electricity

● Quick or immediate results

● Local Materials used whenever possible**

+

● PID Controller:○ must not deviate more than 0.5

Celsius from the set temperature once it is reached

○ heat evenly throughout the resistor

○ minimize overshoot

● Be a more accurate TB diagnostic tool than the ones currently available

○ sensitivity & specificity >= 95%

● Process patient sample in 1-2 hours

● 0.22 mm width with depth of 0.1 mm

● Cost < $9 per test

Specifications

[13]

[10]

Design● Glass chip with etched channels

o Smooth curves

● Silicon Bottomo High Conductivity

● 3 Thin-film Platinum heaterso PID Controlled

● Cooling element unnecessary

Inlet

OutletTop View

Thin FIlm Platinum Heaters/Sensors

Cross Section

Etched Glass LayerSilicon Layer

How it Works

1. Sample with primer, nucleotides, and polymerase is fed into inlet2. Enters Denaturation region ~ 95 C3. Enters Annealing region ~68 C4. Enters Elongation region ~72 C5. Repeat

4

3

2

5

1

PID Control

● Run current through temperature sensitive platinum filmo Temperature detected as voltage drop

● PID controller finds error between set temperature and detected temperature

● Converts error to duty cycle in heating film

● For quick response: kp = high● For stability: ki = low, kd = high

[14]

PID Control: Results● kp = 300, ki = 1, kd = 20● Operating range not as narrow as desired

o Instrument limitations● Overshoot and the steady state error reported 0.7 ◦C and ±0.1 ◦C,

respectively

User Input: 28 COperating Range: 27.5 - 29.5 C

User Input: 50 COperating Range: 48.5 - 52

Building and Testing Prototype

[11]

Testing● Perform PCR with conventional methods and with chip then compare

results

Criteria for Success● Reaction time must be shorter than with conventional method (~2.5 hrs)● Operating range of +/- 0.5 C● Cheap to make/use (< $9)● High Specificity

o Prevent false negatives

Future Work● Address contamination by

adding additional purificationchip

● Test on other disease o viral, genetic, etc.

● Increase scale of productiono Plasmid production

● Test with better instruments withhigher sampling frequency

References[1] "Global Tuberculosis Report 2014." WHO. Web. 7 Mar. 2015.

[2] TB Facts." Team:Paris Bettencourt/Human Practice/TB Fact. IGEM. Web. 7 Mar. 2015.

[3] "The New Worldmapper." Worldmapper: The World as You've Never Seen It before. The University of Sheffield, 3 Jan. 2012. Web. 6 Mar. 2015.

[4] Yang S and Rothman RE. PCR-Based Diagnostics for infectious disease: uses, limitations, and future applications in acute-care settings.

[5] Mueller DH, Mwenge L, Muyoyeta M, et al. “Costs and cost-effectiveness of tuerculosis cultures using solid and liquid media in a developing country.” International Journal of Tuberculosis and Lung Disease. 2008 Oct; 12(10):1196-202.

[6] de Perio MA, Tsewat J, et al. “Cost effectiveness of interferon gamma release assays vs. tuberculin skin tests in health care workers.” JAMA Internal Medicine. 2009 Jan 26;169(2):179-87.

[7] Scherer LC, Sperhacke RD, Ruffino-Netto A, et al. “Cost-effectiveness analysis of PCR for the rapid diagnosis of pulmonary tuberculosis.” BioMed Central Infectious Diseases. 2009, 9:216.

[8] Salian NV, Rish JA, Eisenach KD, et al. “Polymerase chain reaction to detect Mycobacterium tuberculosis in histologic specimens.” American Journal of Respiratory and Critical Care Medicine 1998. Oct;158(4):1150-5.

[9] Shin YS, Cho K, Lim SH, et al. PDMS-based micro PCR chip with Parylene coating. Journal of Micromechanics and microengineering. 2003. June;13:(5):768-774.

[10] Kim J, Byun D, Mauk MG, et al. “A Disposable, Self-Contained PCR Chip.” Lab Chip. 2009 Feb 21; 9(4):606-612.

References (continued)[11] Schneegass I, Brautigam R, Kohler JM. “Miniaturized flow-through PCR with different template types in a silicon chip thermocycler. Lab Chip. 2001 Sep;1(1):42-9.

[12] Oblath EA, Henley WH, Alarie JP, et al. “A microfluidic chip integrating DNA extraction and real time PCR for the detection of bacteria in saliva. Lab Chip. 2013 Apr 7;13(7):1325-32.

[13] Yoon DS, Lee YS, Lee Y, et al. “Precise temperature control and rapid thermal cycling in a micro-machined DNA polymerase chain reaction chip.” Journal of Micromechanics and Microengineering. 2002 Oct; 12(6): 813-823.

[14] "PID Control." PidHingeController. Tin-man, 5 Apr. 2012. Web. 6 Mar. 2015. <https://code.google.com/p/tin-man/wiki/PidHingeController>.