8/20/2019 Application of PCR in African Trypanosomias Field Diagnosis

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Application of PCR in African Trypanosomias field diagnosis

INTRODUCTION

Background

• Sleeping sickness occurs in 36 sub-Saharan Africa

countries where there are tsetse flies that transmit the

disease.

• Human African trypanosomiasis takes 2 forms,

depending on the parasite involved: Trypanosoma

 brucei rhodesiense, found in East Africa, and

Trypanosoma brucei gambiense, which accounts for

more than 98% of reported cases, found in West and

Central Africa.

• The parasite load in T. b. rhodesiense infection is

substantially higher than the level in T. b. gambiense

infection. T.b.rhodiense can be detected through

microscopic detection in blood and cerebral fluid.

• For T.b. gambiense infections, screening for

 potential infection includes serological tests and

checks for clinical signs - especially swollen cervical

lymph nodes.

• The long, relatively asymptomatic first stage of T. b.

gambiense sleeping sickness requires an exhaustive,

active screening of the population at risk

Objective

• Evaluate the accuracy of PCR in gambiense Sleeping

Sickness diagnosis and the benefits of molecular

diagnostics for the patient.

• Develop a standardized PCR diagnosis in field

diagnosis of Sleeping Disease

By Linh Bui

METHODS

• Body Design: Machine the center of the block so that the resistors

can fit in to surroundthe samples. The outside of the block is cut

like a heatsink to allow for faster cool-down.  Diagnosis standardization:

• An 18S ribosomal RNA gene targeting PCR was performed on

 blood and cerebrospinal fluid (CSF) of 360 T. brucei gambiense

sleeping sickness patients and on blood of 129 endemic controlsfrom the Democratic Republic of Congo. Sensitivity and

specificity (with 95% confidence intervals) of PCR for diagnosis,

disease staging and treatment failure over 2 years follow-up post-

treatment were determined.

• PCR:

o The 25 µL reaction mixture contains 1× PCR buffer (Qiagen), 2.5

mM MgCl2 (Qiagen), 200 µM of each dNTP (Roche), 0.8 µM of

sense primer M18S-II-F-Tb (5′-

CGTAGTTGAACTGTGGGCCACGT-3′) (Sigma), 0.8 µM of

antisense primer M18S-II-R-Tb (5′-

ATGCATGACATGCGTGAAAGTGAG- 3′) (Sigma), 2.5 µg

acetylated bovine serum albumin (Promega), 0.5 unit of HotStar

Taq polymerase (Qiagen) and 2.5 µl of specimen DNA

o An initial denaturation step of 94°C for 15 minutes to activate the

HotStar Taq polymerase was followed by 40 cycles of 94°C for 30

seconds, 60°C for 30 seconds and 72°C for 30 seconds and a final

elongation step at 72°C for 5 minutes.

o Amplified products are analysed by electrophoresis in a 2%

agarose gel (Eurogentec) and U.V. illuminated (Syngene) after

ethidium bromide staining (Sigma).

RESULTS

Phase 1:

• Summary sensitivity for PCR on blood was 99.0% (95% CI 92.8 to

99.9) and the specificity was 97.7% (95% CI 93.0 to 99.3).

•  Differences in study design and readout method did not

significantly change estimates although use of satellite DNA as atarget significantly lowers specificity.

• Sensitivity and specificity of PCR on CSF for staging varied from

87.6% to 100%, and 55.6% to 82.9% respectively.

CONCLUSIONS 

• For T.b. gambiense sleeping sickness diagnosis and staging,

PCR performed better than, or similar to, the current parasite

detection techniques but it cannot be used for post-treatment

follow-up

• PCR seems to have sufficient accuracy to replace

microscopy where facilities allow, and can be widely used

with affordable, easily accessible equipment

Phase 1: Evaluation

• Searching, Selection and Data Abstraction:

o Study selection was conducted by two authors (CM and EA)

independently, in the case of disagreements a third author (either

KB or ML) acted as a mediator.

• Quantitative Data Synthesis

o Studies that evaluated the diagnostic value of the tests wereanalyzed separately from studies that evaluated the staging value

of the tests.

o Summary estimates of sensitivity and specificity for diagnosis and

staging for the different assays were calculated.

o Meta-analysis was performed if at least three studies evaluated the

same assay in the same sample type (either blood or CSF)

Phase 2: Integration

Affordable PCR thermocycler design

• Materials: 2 Wiremound resistors, 150 ohms/50 Watts each (10$

total), Arctic Silver Thermal Epoxy (14$), Solid state relay, such as25A AC/DC SSR ($8.50), Aluminum block (64mm x 64mm x

26mm), Arduino board mini ($20), MAX31855 breakout,

Thermocouple wire ($10), 60mm fan ($4), 12V transistor ($0.70),

12DC, 0.5A power supply ($1).

•Circuit design:

• Programming basics: Pulse current across the resistors to heat up

(by setting Arduino pin 7 to high); and turn on the fan to cool down

(by setting Arduino pin 9 to high). Check the temperature by

 polling the thermocouple.

REFERENCES

Deborggraeve, S.,Lejon, V.,Ekangu, R.,Ngoyi, D., Pyana, P.,Ilunga, M.,. . . Büscher, P. (2011). Diagnostic Accuracy ofPCRin

gambiense Sleeping Sickness Diagnosis, Staging and Post-TreatmentFollow-Up:A2-yearLongitudinalStudy. PLoS NeglectedTropical

Diseases PLoS Negl TropDis.

Mugasa, C.,Adams, E.,Boer, K., Dyserinck, H.,Büscher, P.,Schallig, H.,& Leeflang, M. (2012). Diagnostic Accuracy ofMolecular

AmplificationTests forHumanAfricanTrypanosomiasis — Systematic Review. PLoS NeglectedTropical Diseases PLoS Negl TropDis.

Chappuis, F.,Loutan, L.,Simarro, P.,Lejon, V.,& Buscher, P. (2005). Options forFieldDiagnosisof HumanAfricanTrypanosomiasis.

Clinical MicrobiologyReviews, 133-146.

Njiru, Z.,Traub, R.,Ouma, J.,Enyaru, J., &Matovu, E. (2011). DetectionofGroup1 Trypanosoma brucei gambiense by Loop-MediatedIsothermal Amplification. Journal ofClinical Microbiology, 1530-1536.

Phase 2:

•The resulting PCR thermocycler costunder $85 and can get an accuracy of +/-

0.5°C

Diagnosis standardization results:

• The PCR detected 1 parasite in a 180 µl blood sample while

non-spiked control blood samples remained negative. T. b.

gambiense and T. b. rhodesiense DNA was detected at 1 ng

 per test while purified DNA from the non-target pathogens at

50 ng per test did not generate a positive PCR signal.

• Specificity of PCR on blood (PCR-blood) was 99.2% and the

sensitivity on primary HAT cases was 88.4% .The sensitivity

was not significantly different between primary stage 1 and 2 patients (p = 0.184), but was significantly lower in

retreatment cases (p