Application of PCR in African Trypanosomias field diagnosis

By Linh Bui

INTRODUCTION
Background
Sleeping sickness occurs in 36 sub-Saharan Africa
countries where there are tsetse flies that transmit the
disease.

METHODS
Phase 1: Evaluation

Searching, Selection and Data Abstraction:

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.

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

mM MgCl2 (Qiagen), 200 M of each dNTP (Roche), 0.8 M of
sense primer M18S-II-F-Tb (5CGTAGTTGAACTGTGGGCCACGT-3) (Sigma), 0.8 M of
antisense primer M18S-II-R-Tb (5ATGCATGACATGCGTGAAAGTGAG-3) (Sigma), 2.5 g
acetylated bovine serum albumin (Promega), 0.5 unit of HotStar
Taq polymerase (Qiagen) and 2.5 l of specimen DNA

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

Quantitative Data Synthesis

o Studies that evaluated the diagnostic value of the tests were

analyzed 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

Objective

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

total), Arctic Silver Thermal Epoxy (14$), Solid state relay, such as
25A 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:

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

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 controls
from 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 posttreatment were determined.
PCR:

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.

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.

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:

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

HotStar Taq polymerase was followed by 40 cycles of 94C for 30
seconds, 60C for 30 seconds and 72C for 30 seconds and a final
elongation step at 72C 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 a
target significantly lowers specificity.

Phase 2:
The resulting PCR thermocycler cost
under $85 and can get an accuracy of +/0.5C

Diagnosis standardization results:

The PCR detected 1 parasite in a 180 l 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<0.001).
The specificity of PCR on CSF (PCR-CSF) for disease
staging was 82.9% while the sensitivity was 88.4%. There
was no difference in sensitivity of PCR-CSF between
primary and retreatment second stage patients (p = 0.559).
In cured patients, the specificity of PCR-blood was 80.5% 3
months post-treatment, remained stable at a level of 89.9
92.4% between 6 and 18 months post-treatment to slightly
decrease to 81.2% 24 months post-treatment. Between 12 and
24 months after treatment, 24.6% (95% CI 18.131.0) of
cured patients had at least 1 PCR-blood positive result.
In cured patients, the specificity of PCR-CSF slowly
increased from 56.3% at 3 months after treatment to stagnate
between 79.7 and 83.7% from 12 months post-treatment until
the end of follow-up. Between 12 and 24 months post
treatment, 35.4% (95% CI 28.342.6) of cured patients were
CSF PCR positive on at least one occasion when tested.

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

Sensitivity and specificity of PCR on CSF for staging varied from

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

REFERENCES
Deborggraeve, S., Lejon, V., Ekangu, R., Ngoyi, D., Pyana, P., Ilunga, M., . . . Bscher, P. (2011). Diagnostic Accuracy of PCR in
gambiense Sleeping Sickness Diagnosis, Staging and Post-Treatment Follow-Up: A 2-year Longitudinal Study. PLoS Neglected Tropical
Diseases PLoS Negl Trop Dis.

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.

Mugasa, C., Adams, E., Boer, K., Dyserinck, H., Bscher, P., Schallig, H., & Leeflang, M. (2012). Diagnostic Accuracy of Molecular
Amplification Tests for Human African TrypanosomiasisSystematic Review. PLoS Neglected Tropical Diseases PLoS Negl Trop Dis.
Chappuis, F., Loutan, L., Simarro, P., Lejon, V., & Buscher, P. (2005). Options for Field Diagnosis of Human African Trypanosomiasis.
Clinical Microbiology Reviews, 133-146.
Njiru, Z., Traub, R., Ouma, J., Enyaru, J., & Matovu, E. (2011). Detection of Group 1 Trypanosoma brucei gambiense by LoopMediated Isothermal Amplification. Journal of Clinical Microbiology, 1530-1536.